Methods for producing multivalent antigen-binding proteins

ABSTRACT

Compositions of, genetic constructions coding for, and methods for producing multivalent antigen-binding proteins are described and claimed. The methods include purification of compositions containing both monomeric and multivalent forms of single polypeptide chain molecules, and production of multivalent proteins from purified monomers. Production of multivalent proteins may occur by a concentration-dependent association of monomeric proteins, or by rearrangement of regions involving dissociation followed by reassociation of different regions. Bivalent proteins, including homobivalent and heterobivalent proteins, are made in the present invention. Genetic sequences coding for bivalent single-chain antigen-binding proteins are disclosed. Uses include all those appropriate for monoclonal and polyclonal antibodies and fragments thereof, including use as a bispecific antigen-binding molecule.

This invention was made with Government Support under SBIR Grant 5R44 GM 39662-03 awarded by the National Institutes of Health, National Institute of General Medical Sciences. The Government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 08/392,338, filed Feb. 22, 1995, issued as U.S. Pat. No. 5,869,620; which is a divisional of U.S. patent application Ser. No. 07/989,846, filed Nov. 20, 1992, now abandoned; which is a continuation-in-part of U.S. patent application Ser. No. 07/796,936, filed Nov. 25, 1991, now abandoned. The contents of each of the above mentioned applications are fully incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the production of antigen-binding molecules. More specifically, the invention relates to multivalent forms of antigen-binding proteins. Compositions of, genetic constructions for, methods of use, and methods for producing these multivalent antigen-binding proteins are disclosed.

2. Desciption of the Background Art

Antibodies are proteins generated by the immune system to provide a specific molecule capable of complexing with an invading molecule, termed an antigen. FIG. 14 shows the structure of a typical antibody molecule. Natural antibodies have two identical antigen-binding sites, both of which are specific to a particular antigen. The antibody molecule "recognizes" the antigen by complexing its antigen-binding sites with areas of the antigen termed epitopes. The epitopes fit into the conformational architecture of the antigen-binding sites of the antibody, enabling the antibody to bind to the antigen.

The antibody molecule is composed of two identical heavy and two identical light polypeptide chains, held together by interchain disulfide bonds (see FIG. 14). The remainder of this discussion will refer only to one light/heavy pair of chains, as each light/heavy pair is identical. Each individual light and heavy chain folds into regions of approximately 110 amino acids, assuming a conserved three-dimensional conformation. The light chain comprises one variable region (termed V_(L)) and one constant region (C_(L)), while the heavy chain comprises one variable region (V_(H)) and three constant regions (C_(H) 1, C_(H) 2 and C_(H) 3). Pairs of regions associate to form discrete structures as shown in FIG. 14. In particular, the light and heavy chain variable regions, V_(L) and V_(H), associate to form an "F_(V) " area which contains the antigen-binding site.

The variable regions of both heavy and light chains show considerable variability in structure and amino acid composition from one antibody molecule to another, whereas the constant regions show little variability. The term "variable" as used in this specification refers to the diverse nature of the amino acid sequences of the antibody heavy and light chain variable regions. Each antibody recognizes and binds antigen through the binding site defined by the association of the heavy and light chain variable regions into an F_(V) area. The light-chain variable region V_(L) and the heavy-chain variable region V_(H) of a particular antibody molecule have specific amino acid sequences that allow the antigen-binding site to assume a conformation that binds to the antigen epitope recognized by that particular antibody.

Within the variable regions are found regions in which the amino acid sequence is extremely variable from one antibody to another. Three of these so-called "hypervariable" regions or "complementarity-determining regions" (CDR's) are found in each of the light and heavy chains. The three CDR's from a light chain and the three CDR's from a corresponding heavy chain form the antigen-binding site.

Cleavage of the naturally-occurring antibody molecule with the proteolytic enzyme papain generates fragments which retain their antigen-binding site. These fragments, commonly known as Fab's (for Fragment, antigen binding site) are composed of the C_(L), V_(L), C_(H) 1 and V_(H) regions of the antibody. In the Fab the light chain and the fragment of the heavy chain are covalently linked by a disulfide linkage.

Recent advances in immunobiology, recombinant DNA technology, and computer science have allowed the creation of single polypeptide chain molecules that bind antigen. These single-chain antigen-binding molecules incorporate a linker polypeptide to bridge the individual variable regions, V_(L) and V_(H), into a single polypeptide chain. A computer-assisted method for linker design is described more particularly in U.S. Pat. No. 4,704,692, issued to Iadner et al. in November, 1987, and incorporated herein by reference. A description of the theory and production of single-chain antigen-binding proteins is found in U.S. Pat. No. 4,946,778 (Ladner et al.), issued Aug. 7, 1990, and incorporated herein by reference. The single-chain antigen-binding proteins produced under the process recited in U.S. Pat. No. 4,946,778 have binding specificity and affinity substantially similar to that of the corresponding Fab fragment.

Bifunctional, or bispecific, antibodies have antigen binding sites of different specificities. Bispecific antibodies have been generated to deliver cells, cytotoxins, or drugs to specific sites. An important use has been to deliver host cytotoxic cells, such as natural killer or cytotoxic T cells, to specific cellular targets. (U. D. Staerz, O. Kanagawa, M. J. Bevan, Nature 314:628 (1985); S. Songilvilai, P. J. Lachmann, Clin. Exp. Immunol. 79: 315 (1990)). Another important use has been to deliver cytotoxic proteins to specific cellular targets. (V. Raso, T. Griffin, Cancer Res. 41:2073 (1981); S. Honda, Y. Ichimori, S. Iwasa, Cytotechnology 4:59 (1990)). Another important use has been to deliver anti-cancer non-protein drugs to specific cellular targets (J. Corvalan, W. Smith, V. Gore, Intl. J. Cancer Sappl. 2:22 (1988); M. Pimm et al., British J. of Cancer 61:508 (1990)). Such bispecific antibodies have been prepared by chemical cross-linking (M. Brennan et al., Science 229:81 (1985)), disulfide exchange, or the production of hybrid-hybridomas (quadromas). Quadromas are constructed by fusing hybridomas that secrete two different types of antibodies against two different antigens (Kurokawa, T. et al., Biotechnology 7.1163 (1989)).

SUMMARY OF THE INVENTION

This invention relates to the discovery that multivalent forms of single-chain antigen-binding proteins have significant utility beyond that of the monovalent single-chain antigen-binding proteins. A multivalent antigen-binding protein has more than one antigen-binding site. Enhanced binding activity, di- and multi-specific binding, and other novel uses of multivalent antigen-binding proteins have been demonstrated or are envisioned here. Accordingly, the invention is directed to multivalent forms of single-chain antigen-binding proteins, compositions of multivalent and single-chain antigen-binding proteins, methods of making and purifying multivalent forms of single-chain antigen-binding proteins, and uses for multivalent forms of single-chain antigen-binding proteins. The invention provides a multivalent antigen-binding protein comprising two or more single-chain protein molecules, each single-chain molecule comprising a first polypeptide comprising the binding portion of the variable region of an antibody heavy or light chain; a second polypeptide comprising the binding portion of the variable region of an antibody heavy or light chain; and a peptide linker linking the first and second polypeptides into a single-chain protein.

Also provided is a composition comprising a multivalent antigen-binding protein substantially free of single-chain molecules.

Also provided is an aqueous composition comprising an excess of multivalent antigen-binding protein over single-chain molecules.

A method of producing a multivalent antigen-binding protein is provided, comprising the steps of producing a composition comprising multivalent antigen-binding protein and single-chain molecules, each single-chain molecule comprising a first polypeptide comprising the binding portion of the variable region of an antibody heavy or light chain; a second polypeptide comprising the binding portion of the variable region of an antibody heavy or light chain; and a peptide linker linking the first and second polypeptides into a single-chain molecule; separating the multivalent protein from the single-chain molecules; and recovering the multivalent protein.

Also provided is a method of producing multivalent antigen-binding protein, comprising the steps of producing a composition comprising single-chain molecules as previously defined; dissociating the single-chain molecules; reassociating the single-chain molecules; separating the resulting multivalent antigen-binding proteins from the single-chain molecules; and recovering the multivalent proteins.

Also provided is another method of producing a multivalent antigen-binding protein, comprising the step of chemically cross-linking at least two single-chain antigen-binding molecules.

Also provided is another method of producing a multivalent antigen-binding protein, comprising the steps of producing a composition comprising single-chain molecules as previously defined; concentrating said single-chain molecules; separating said multivalent protein from said single-chain molecules; and finally recovering said multivalent protein.

Also provided is another method of producing a multivalent antigen-binding protein comprising two or more single-chain molecules, each single-chain molecule as previously defined, said method comprising: providing a genetic sequence coding for said single-chain molecule; transforming a host cell or cells with said sequence; expressing said sequence in said host or hosts; and recovering said multivalent protein.

Another aspect of the invention includes a method of detecting an antigen in or suspected of being in a sample, which comprises contacting said sample with the multivalent antigen-binding protein of claim 1 and detecting whether said multivalent antigen-binding protein has bound to said antigen.

Another aspect of the invention includes a method of imaging the internal structure of an animal, comprising administering to said animal an effective amount of a labeled form of the multivalent antigen-binding protein of claim 1 and measuring detectable radiation associated with said animal.

Another aspect of the invention includes a composition comprising an association of a multivalent antigen-binding protein with a therapeutically or diagnostically effective agent.

Another aspect of this invention is a single-chain protein comprising: a first polypeptide comprising the binding portion of the variable region of an antibody light chain; a second polypeptide comprising the binding portion of the variable region of an antibody light chain; a peptide linker linking said first and second polypeptides (a) and (b) into said single-chain protein.

Another aspect of the present invention includes the genetic constructions encoding the combinations of regions V_(L) --V_(L) and V_(H) --V_(H) for single-chain molecules, and encoding multivalent antigen-binding proteins.

Another part of this invention is a multivalent single-chain antigen-binding protein comprising: a first polypeptide comprising the binding portion of the variable region of an antibody heavy or light chain; a second polypeptide comprising the binding portion of the variable region of an antibody heavy or light chain; a peptide linker linking said first and second polypeptides (a) and (b) into said multivalent protein; a third polypeptide comprising the binding portion of the variable region of an antibody heavy or light chain; a fourth polypeptide comprising the binding portion of the variable region of an antibody heavy or light chain; a peptide linker linking said third and fourth polypeptides (d) and (e) into said multivalent protein; and a peptide linker linking said second and third polypeptides (b) and (d) into said multivalent protein. Also included are genetic constructions coding for this multivalent single-chain antigen-binding protein.

Also included are replicable cloning or expression vehicles including plasmids, hosts transformed with the aforementioned genetic sequences, and methods of producing multivalent proteins with the sequences, transformed hosts, and expression vehicles.

Methods of use are provided, such as a method of using the multivalent antigen-binding protein to diagnose a medical condition; a method of using the multivalent protein as a carrier to image the specific bodily organs of an animal; a therapeutic method of using the multivalent protein to treat a medical condition; and an immunotherapeutic method of conjugating a multivalent protein with a therapeutically or diagnostically effective agent. Also included are labelled multivalent proteins, improved immunoassays using them, and improved immunoaffinity purifications.

An advantage of using multivalent antigen-binding proteins instead of single-chain antigen-binding molecules or Fab fragments lies in the enhanced binding ability of the multivalent form. Enhanced binding occurs because the multivalent form has more binding sites per molecule. Another advantage of the present invention is the ability to use multivalent antigen-binding proteins as multi-specific binding molecules.

An advantage of using multivalent antigen-binding proteins instead of whole antibodies, is the enhanced clearing of the multivalent antigen-binding proteins from the serum due to their smaller size as compared to whole antibodies which may afford lower background in imaging applications. Multivalent antigen-binding proteins may penetrate solid tumors better than monoclonals, resulting in better tumor-fighting ability. Also, because they are smaller and lack the Fc component of intact antibodies, the multivalent antigen-binding proteins of the present invention may be less immunogenic than whole antibodies. The Fc component of whole antibodies also contains binding sites for liver, spleen and certain other cells and its absence should thus reduce accumulation in non-target tissues.

Another advantage of multivalent antigen-binding proteins is the ease with which they may be produced and engineered, as compared to the myeloma-fusing technique pioneered by Kohler and Milstein that is used to produce whole antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention as defined in the claims can be better understood with reference to the text and to the following drawings:

FIG. 1A is a schematic two-dimensional representation of two identical single-chain antigen-binding protein molecules, each comprising a variable light chain region (V_(L)), a variable heary chain region (V_(H)), and a polypeptide linker joining the two regions. The single-chain antigen-binding protein molecules are shown binding antigen in their antigen-binding sites.

FIG. 1B depicts a hypothetical homodivalent antigen-binding protein formed by association of the polypeptide linkers of two monovalent single-chain antigen-binding proteins from FIG. 1A (the Association model). The divalent antigen-binding protein is formed by the concentration-driven association of two identical single-chain antigen-binding protein molecules.

FIG. 1C depicts the hypothetical divalent protein of FIG. 1B with bound antigen molecules occupying both antigen-binding sites.

FIG. 2A depicts the hypothetical homodivalent protein of FIG. 1B.

FIG. 2B depicts three single-chain antigen-binding protein molecules associated in a hypothetical trimer.

FIG. 2C depicts a hypothetical tetramer of four single-chain antigen-binding protein molecules.

FIG. 3A depicts two separate and distinct monovalent single-chain antigen-binding proteins, Anti-A single-chain antigen-binding protein and Anti-B single-chain antigen-binding protein, with different antigen specificities, each individually binding either Antigen A or Antigen B.

FIG. 3B depicts a hypothetical bispecific heterodivalent antigen-binding protein formed from the single-chain antigen-binding proteins of FIG. 3A according to the Association model.

FIG. 3C depicts the hypothetical heterodivalent antigen-binding protein of FIG. 3B binding bispecifically, i.e., binding the two different antigens, A and B.

FIG. 4A depicts two identical single-chain antigen-binding protein molecules, each having a variable light chain region (V_(L)), a variable heavy chain region (V_(H)), and a polypeptide linker joining the two regions. The single-chain antigen-binding protein molecules are shown binding identical antigen molecules in their antigen-binding sites.

FIG. 4B depicts a hypothetical homodivalent protein formed by the rearrangement of the V_(L) and V_(H) regions shown in FIG. 4A (the Rearrangement model). Also shown is bound antigen.

FIG. 5A depicts two single-chain protein molecules, the first having an anti-B V_(L) and an anti-A V_(H), and the second having an anti-A V_(L) and an anti-B V_(H). The figure shows the non-complementary nature of the V_(L) and V_(H) regions in each single-chain protein molecule.

FIG. 5B shows a hypothetical bispecific heterodivalent antigen-binding protein formed by rearrangement of the two single-chain proteins of FIG. 5A.

FIG. 5C depicts the hypothetical heterodivalent antigen-binding protein of FIG. 5B with different antigens A and B occupying their respective antigen-binding sites.

FIG. 6A is a schematic depiction of a hypothetical trivalent antigen-binding protein according to the Rearrangement model.

FIG. 6B is a schematic depiction of a hypothetical tetravalent antigen-binding protein according to the Rearrangement model.

FIG. 7 is a chromatogram depicting the separation of CC49/212 antigen-binding protein monomer from dimer on a cation exchange high performance liquid chromatographic column. The column is a PolyCAT A aspartic acid column (Poly WC, Columbia, Md.). Monomer is shown as Peak 1, eluting at 27.32 min., and dimer is shown as Peak 2, eluting at 55.52 min.

FIG. 8 is a chromatogram of the purified monomer from FIG. 7. Monomer elutes at 21.94 min., preceded by dimer (20.135 min.) and trimer (18.640 min.). Gel filtration column, Protein-Pak 300SW (Waters Associates, Milford, Mass.).

FIG. 9 is a similar chromatogram of purified dimer (20.14 min.) from FIG. 7, run on the gel filtration HPLC column of FIG. 8.

FIG. 10A is an amino acid (SEQ ID NO. 11) and nucleotide (SEQ ID NO. 10) sequence of the single-chain protein comprising the 4-4-20 V_(L) region connected through the 212 linker polypeptide to the CC49 V_(H) region.

FIG. 10B is an amino acid (SEQ ID NO. 13) and nucleotide (SEQ ID NO. 12) sequence of the single-chain protein comprising the CC49 V_(L) region connected through the 212 linker polypeptide to the 4-4-20 V_(H) region.

FIG. 11 is a chromatogram depicting the separation of the monomer (27.83 min.) and dimer (50.47 min.) forms of the CC49/212 antigen-binding protein by cation exchange, on a PolyCAT A cation exchange column (Poly LC, Columbia, Md.).

FIG. 12 shows the separation of monomer (17.65 min.), dimer (15.79 min.), trimer (14.19 min.), and higher oligomers (shoulder at about 13.09 min.) of the B6.2/212 antigen-binding protein. This separation depicts the results of a 24-hour treatment of a 1.0 mg/ml B6.2/212 single-chain antigen-binding protein sample. A TSK G2000SW gel filtration HPLC column was used, Toyo Soda, Tokyo, Japan.

FIG. 13 shows the results of a 24-hour treatment of a 4.0 mg/ml CC49/212 antigen-binding protein sample, generating monomer, dimer, and trimer at 16.91, 14.9, and 13.42 min., respectively. The same TSK gel filtration column was used as in FIG. 12.

FIG. 14 shows a schematic view of the four-chain structure of a human IgG molecule.

FIG. 15A is an amino acid (SEQ ID NO. 15) and nucleotide (SEQ ID NO. 14) sequence of the 4-4-20/212 single-chain antigen-binding protein with a single cysteine hinge.

FIG. 15B is an amino acid (SEQ ID NO. 17) and nucleotide (SEQ. ID NO. 16) sequence of the 4-4-20/212 single-chain antigen-binding protein with the two-cysteine hinge.

FIG. 16 shows the amino acid (SEQ ID NO. 19) and nucleotide (SEQ ID NO. 18) sequence of a divalent CC49/212 single-chain antigen-binding protein.

FIG. 17 shows the expression of the divalent CC49/212 single-chain antigen-binding protein of FIG. 16 at 42° C., on an SDS-PAGE gel containing total E. coli protein. Lane 1 contains the molecular weight standards. Lane 2 is the uninduced E. coli production strain grown at 30° C. Lane 3 is divalent CC49/212 single-chain antigen-binding protein induced by growth at 42° C. The arrow shows the band of expressed divalent CC49/212 single-chain antigen-binding protein.

FIG. 18 is a graphical representation of four competition radioimmunoassays (RIA) in which unlabeled CC49 IgG (open circles) CC49/212 single-chain antigen-binding protein (closed circles) and CC49/212 divalent antigen-binding protein (closed squares) and anti-fluorescein 4-4-20/212 single-chain antigen-binding protein (open squares) competed against a CC49 IgG radiolabeled with ¹²⁵ I for binding to the TAG-72 antigen on a human breast carcinoma extract.

FIG. 19A is an amino acid (SEQ ID NO. 21) and nucleotide (SEQ ID NO. 20) sequence of the single-chain polypeptide comprising the 4-4-20 V_(L) region connected through the 217 linker polypeptide to the CC49 V_(H) region.

FIG. 19B is an amino acid (SEQ ID NO. 23) and nucleotide (SEQ ID NO. 22) sequence of the single-chain polypeptide comprising the CC49 V_(L) region connected through the 217 linker polypeptide to the 4-4-20 V_(H) region.

FIG. 20 is a chromatogram depicting the purification of CC49/4-4-20 heterodimer Fv on a cation exchange high performance liquid chromatographic column. The column is a PolyCAT A aspartic acid column (Poly LC, Columbia, Md.). The heterodimer Fv is shown as fraction 5, eluting at 30.10 min.

FIG. 21 is a coomassie-blue stained 4-20% SDS-PAGE gel showing the proteins separated in FIG. 20. Lane 1 contains the molecular weight standards. Lane 3 contains the starting material before separation. Lanes 4-8 contain fractions 2, 3, 5, 6 and 7 respectively. Lane 9 contains purified CC49/212.

FIG. 22A is a chromatogram used to determine the molecular size of fraction 2 from FIG. 20. A TSK G3000SW gel filtration HPLC column was used (Toyo Soda, Tokyo, Japan).

FIG. 22B is a chromatogram used to determine the molecular size of fraction 5 from FIG. 20. A TSK G3000SW gel filtration HPLC column was used (Toyo Soda, Tokyo, Japan).

FIG. 22C is a chromatogram used to determine the molecular size of fraction 6 from FIG. 20. A TSK G30005W gel filtration HPLC column was used (Toyo Soda, Tokyo, Japan).

FIG. 23 shows a Scatchard analysis of the fluorescein binding affinity of the CC49 4-4-20 heterodimer F_(V) (fraction 5 in FIG. 20).

FIG. 24 is a graphical representation of three competition enzyme-linked immunosorbent assays (ELISA) in which unlabeled CC49 4-4-20 Fv (closed squares) CC49/212 single-chain Fv (open squares) and MOPC-21 IgG (+) competed against a biotin-labeled CC49 IgG for binding to the TAG-72 antigen on a human breast carcinoma extract. MOPC-21 is a control antibody that does not bind to TAG-72 antigen.

FIG. 25 shows a coomassie-blue stained non-reducing 4-20% SDS-PAGE gel. Lanes 1 and 9 contain the molecular weight standards. Lane 3 contains the 4-4-20/212 CPPC single-chain antigen-binding protein after purification. Lane 4, 5 and 6 contain the 4-4-20/212 CPPC single-chain antigen-binding protein after treatment with DTT and air oxidation. Lane 7 contains 4-4-20/212 single-chain antigen-binding protein.

FIG. 26 shows a coomassie-blue stained reducing 4-20% SDS-PAGE gel (samples were treated with β-mercaptoethanol prior to being loaded on the gel). Lanes 1 and 8 contain the molecular weight standards. Lane 3 contains the 4-4-20/212 CPPC single-chain antigen-binding protein after treatment with bis-maleimidehexane. Lane 5 contains peak 1 of bis-maleimidehexane treated 4-4-20/212 CPCC single-chain antigen-binding protein. Lane 6 contains peak 3 of bis-maleimidehexane treated 4-4-20/212 CPPC single-chain antigen-binding protein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention relates to the discovery that multivalent forms of single-chain antigen-binding proteins have significant utility beyond that of the monovalent single-chain antigen-binding proteins. A multivalent antigen-binding protein has more than one antigen-binding site. For the purposes of this application, "valent" refers to the numerosity of antigen binding sites. Thus, a bivalent protein refers to a protein with two binding sites. Enhanced binding activity, bi- and multi-specific binding, and other novel uses of multivalent antigen-binding proteins have been demonstrated or are envisioned here. Accordingly, the invention is directed to multivalent forms of single-chain antigen-binding proteins, compositions of multivalent and single-chain antigen-binding proteins, methods of making and purifying multivalent forms of single-chain antigen-binding proteins, and new and improved uses for multivalent forms of single-chain antigen-binding proteins. The invention provides a multivalent antigen-binding protein comprising two or more single-chain protein molecules, each single-chain molecule comprising a first polypeptide comprising the binding portion of the variable region of an antibody heavy or light chain; a second polypeptide comprising the binding portion of the variable region of an antibody heavy or light chain; and a peptide linker linking the first and second polypeptides into a single-chain protein.

The term "multivalent" means any assemblage, covalently or non-covalently joined, of two or more single-chain proteins, the assemblage having more than one antigen-binding site. The single-chain proteins composing the assemblage may have antigen-binding activity, or they may lack antigen-binding activity individually but be capable of assembly into active multivalent antigen-binding proteins. The term "multivalent" encompasses bivalent, trivalent, tetravalent, etc. It is envisioned that multivalent forms above bivalent may be useful for certain applications.

A preferred form of the multivalent antigen-binding protein comprises bivalent proteins, including heterobivalent and homobivalent forms. The term "bivalent" means an assemblage of single-chain proteins associated with each other to form two antigen-binding sites. The term "heterobivalent" indicates multivalent antigen-binding proteins that are bispecific molecules capable of binding to two different antigenic determinants. Therefore, heterobivalent proteins have two antigen-binding sites that have different binding specificities. The term "homobivalent" indicates that the two binding sites are for the same antigenic determinant.

The terms "single-chain molecule" or "single-chain protein" are used interchangeably here. They are structurally defined as comprising the binding portion of a first polypeptide from the variable region of an antibody, associated with the binding portion of a second polypeptide from the variable region of an antibody, the two polypeptides being joined by a peptide linker linking the first and second polypeptides into a single polypeptide chain. The single polypeptide chain thus comprises a pair of variable regions connected by a polypeptide linker. The regions may associate to form a functional antigen-binding site, as in the case wherein the regions comprise a light-chain and a heavy-chain variable region pair with appropriately paired complementarity determining regions (CDRs). In this case, the single-chain protein is referred to as a "single-chain antigen-binding protein" or "single-chain antigen-binding molecule."

Alternatively, the variable regions may have unnaturally paired CDRs or may both be derived from the same kind of antibody chain, either heavy or light, in which case the resulting single-chain molecule may not display a functional antigen-binding site. The single-chain antigen-binding protein molecule is more fully described in U.S. Pat. No. 4,946,778 (Ladner et al.), and incorporated herein by reference.

Without being bound by any particular theory, the inventors speculate on several models which can equally explain the phenomenon of multivalence. The inventors' models are presented herein for the purpose of illustration only, and are not to be construed as limitations upon the scope of the invention. The invention is useful and operable regardless of the precise mechanism of multivalence.

FIG. 1 depicts the first hypothetical model for the creation of a multivalent protein, the "Association" model. FIG. 1A shows two monovalent single-chain antigen-binding proteins, each composed of a V_(L), a V_(H), and a linker polypeptide covalently bridging the two. Each monovalent single-chain antigen-binding protein is depicted having an identical antigen-binding site containing antigen. FIG. 1B shows the simple association of the two single-chain antigen-binding proteins to create the bivalent form of the multivalent protein. It is hypothesized that simple hydrophobic forces between the monovalent proteins are responsible for their association in this manner. The origin of the multivalent proteins may be traceable to their concentration dependence. The monovalent units retain their original association between the V_(H) and V_(L) regions. FIG. 1C shows the newly-formed homobivalent protein binding two identical antigen molecules simultaneously. Homobivalent antigen-binding proteins are necessarily monospecific for antigen.

Homovalent proteins are depicted in FIGS. 2A through 2C formed according to the Association model. FIG. 1A depicts a homobivalent protein, FIG. 2B a trivalent protein, and FIG. 2C a tetravalent protein. Of course, the limitations of two-dimensional images of three-dimensional objects must be taken into account. Thus, the actual spatial arrangement of multivalent proteins can be expected to vary somewhat from these figures.

A heterobivalent antigen-binding protein has two different binding sites, the sites having different binding specificities. FIGS. 3A through C depict the Association model pathway to the creation of a heterobivalent protein. FIG. 3A shows two monovalent single-chain antigen-binding proteins, Anti-A single-chain antigen-binding protein and Anti-B single-chain antigen-binding protein, with antigen types A and B occupying the respective binding sites. FIG. 3B depicts the heterobivalent protein formed by the simple association of the original monovalent proteins. FIG. 3C shows the heterobivalent protein having bound antigens A and B into the antigen-binding sites. FIG. 3C therefore shows the heterobivalent protein binding in a bispecific manner.

An alternative model for the formation of multivalent antigen-binding proteins is shown in FIGS. 4 through 6. This "Rearrangement" model hypothesizes the dissociation of the variable region interface by contact with dissociating agents such as guanidine hydrochloride, urea, or alcohols such as ethanol, either alone or in combination. Combinations and relevant concentration ranges of dissociating agents are recited in the discussion concerning dissociating agents, and in Example 2. Subsequent re-association of dissociated regions allows variable region recombination differing from the starting single-chain proteins, as depicted in FIG. 4B. The homobivalent antigen-binding protein of FIG. 4B is formed from the parent single-chain antigen-binding proteins shown in FIG. 4A, the recombined bivalent protein having V_(L) and V_(H) from the parent monovalent single-chain proteins. The homobivalent protein of FIG. 4B is a fully functional monospecific bivalent protein, shown actively binding two antigen molecules.

FIGS. 5A-5C show the formation of heterobivalent antigen-binding proteins via the Rearrangement model. FIG. 5A shows a pair of single-chain proteins, each having a V_(L) with complementarity determining regions (CDRs) that do not match those of the associated V_(H). These single-chain proteins have reduced or no ability to bind antigen because of the mixed nature of their antigen-binding sites, and thus are made specifically to be assembled into multivalent proteins through this route. FIG. 5B shows the heterobivalent antigen-binding protein formed whereby the V_(H) and V_(L) regions of the parent proteins are shared between the separate halves of the heterobivalent protein. FIG. 5C shows the binding of two different antigen molecules to the resultant functional bispecific heterobivalent protein. The Rearrangement model also explains the generation of multivalent proteins of a higher order than bivalent, as it can be appreciated that more than a pair of single-chain proteins can be reassembled in this manner. These are depicted in FIGS. 6A and 6B.

One of the major utilities of the multivalent antigen-binding protein is in the heterobivalent form, in which one specificity is for one type of hapten or antigen, and the second specificity is for a second type of hapten or antigen. A multivalent molecule having two distinct binding specificities has many potential uses. For instance, one antigen binding site may be specific for a cell-surface epitope of a target cell, such as a tumor cell or other undesirable cell. The other antigen-binding site may be specific for a cell-surface epitope of an effector cell, such as the CD3 protein of a cytotoxic T-cell. In this way, the heterobivalent antigen-binding protein may guide a cytotoxic cell to a particular class of cells that are to be preferentially attacked.

Other uses of heterobivalent antigen-binding proteins are the specific targeting and destruction of blood clots by a bispecific molecule with specificity for tissue plasminogen activator (tPA) and fibrin; the specific targeting of pro-drug activating enzymes to tumor cells by a bispecific molecule with specificity for tumor cells and enzyme; and specific targeting of cytotoxic proteins to tumor cells by a bispecific molecule with specificity for tumor cells and a cytotoxic protein. This list is illustrative only, and any use for which a multivalent specificity is appropriate comes within the scope of this invention.

The invention also extends to uses for the multivalent antigen-binding proteins in purification and biosensors. Affinity purification is made possible by affixing the multivalent antigen-binding protein to a support, with the antigen-binding sites exposed to and in contact with the ligand molecule to be separated, and thus purified. Biosensors generate a detectable signal upon binding of a specific antigen to an antigen-binding molecule, with subsequent processing of the signal. Multivalent antigen-binding proteins, when used as the antigen-binding molecule in biosensors, may change conformation upon binding, thus generating a signal that may be detected.

Essentially all of the uses for which monoclonal or polyclonal antibodies, or fragments thereof, have been envisioned by the prior art, can be addressed by the multivalent proteins of the present invention. These uses include detectably-labelled forms of the multivalent protein. Types of labels are well-known to those of ordinary skill in the art. They include radiolabelling, chemiluminescent labeling, fluorochromic labelling, and chromophoric labeling. Other uses include imaging the internal structure of an animal (including a human) by administering an effective amount of a labelled form of the multivalent protein and measuring detectable radiation associated with the animal. They also include improved immunoassays, including sandwich immunoassay, competitive immunoassay, and other immunoassays wherein the labelled antibody can be replaced by the multivalent antigen-binding protein of this invention.

A first preferred method of producing multivalent antigen-binding proteins involves separating the multivalent proteins from a production composition that comprises both multivalent and single-chain proteins, as represented in Example 1. The method comprises producing a composition of multivalent and single-chain proteins, separating the multivalent proteins from the single-chain proteins, and recovering the multivalent proteins.

A second preferred method of producing multivalent antigen-binding proteins comprises the steps of producing single-chain protein molecules, dissociating said single-chain molecules, reassociating the single-chain molecules such that a significant fraction of the resulting composition includes multivalent forms of the single-chain antigen-binding proteins, separating multivalent antigen-binding proteins from single-chain molecules, and recovering the multivalent proteins. This process is illustrated with more detail in Example 2. For the purposes of this method, the term "producing a composition comprising single-chain molecules" may indicate the actual production of these molecules. The term may also include procuring them from whatever commercial or institutional source makes them available. Use of the term "producing single-chain proteins" means production of single-chain proteins by any process, but preferably according to the process set forth in U.S. Pat. No. 4,946,778 (Ladner et al.). Briefly, that patent pertains to a single polypeptide chain antigen-binding molecule which has binding specificity and affinity substantially similar to the binding specificity and affinity of the aggregate light and heavy chain variable regions of an antibody, to genetic sequences coding therefore, and to recombinant DNA methods of producing such molecules, and uses for such molecules. The single-chain protein produced by the Ladner et aL methodology comprises two regions linked by a linker polypeptide. The two regions are termed the V_(H) and V_(L) regions, each region comprising one half of a functional antigen-binding site.

The term "dissociating said single-chain molecules" means to cause the physical separation of the two variable regions of the single-chain protein without causing denaturation of the variable regions.

"Dissociating agents" are defined herein to include all agents capable of dissociating the variable regions, as defined above. In the context of this invention, the term includes the well-known agents alcohol (including ethanol), guanidine hydrochloride (GuHCl), and urea. Others will be apparent to those of ordinary skill in the art, including detergents and similar agents capable of interrupting the interactions that maintain protein conformation. In the preferred embodiment, a combination of GuHCl and ethanol (EtOH) is used as the dissociating agent. A preferred range for ethanol and GuHCl is from 0 to 50% EtOH, vol/vol, 0 to 2.0 moles per liter (M) GuHCl. A more preferred range is from 10-30% EtOH and 0.5-1.0 M GuHCl, and a most preferred range is 20% EtOH, 0.5 M GuHCl. A preferred dissociation buffer contains 0.5 M guanidine hydrochloride, 20% ethanol, 0.05 M TRIS, and 0.01 M CaCl₂, pH 8.0.

Use of the term "re-associating said single-chain molecules" is meant to describe the reassociation of the variable regions by contacting them with a buffer solution that allows reassociation. Such a buffer is preferably used in the present invention and is characterized as being composed of 0.04 M MOPS, 0.10 M calcium acetate, pH 7.5. Other buffers allowing the reassociation of the V_(L) and V_(H) regions are well within the expertise of one of ordinary skill in the art.

The separation of the multivalent protein from the single-chain molecules occurs by use of standard techniques known in the art, particularly including cation exchange or gel filtration chromatography.

Cation exchange chromatography is the general liquid chromatographic technique of ion-exchange chromatography utilizing anion columns well-known to those of ordinary skill in the art. In this invention, the cations exchanged are the single-chain and multivalent protein molecules. Since multivalent proteins will have some multiple of the net charge of the single-chain molecule, the multivalent proteins are retained more strongly and are thus separated from the single-chain molecules. The preferred cationic exchanger of the present invention is a polyaspartic acid column, as shown in FIG. 7. FIG. 7 depicts the separation of single-chain protein (Peak 1, 27.32 min.) from bivalent protein (Peak 2, 55.54 min.) Those of ordinary skill in the art will realize that the invention is not limited to any particular type of chromatography column, so long as it is capable of separating the two forms of protein molecules.

Gel filtration chromatography is the use of a gel-like material to separate proteins on the basis of their molecular weight. A "gel" is a matrix of water and a polymer, such as agarose or polymerized acrylamide. The present invention encompasses the use of gel filtration HPLC (high performance liquid chromatography), as will be appreciated by one of ordinary skill in the art. FIG. 8 is a chromatogram depicting the use of a Waters Associates' Protein-Pak 300 SW gel filtration column to separate monovalent single-chain protein from multivalent protein, including the monomer (21.940 min.), bivalent protein (20.135 min.), and trivalent protein (18.640 min.).

Recovering the multivalent antigen-binding proteins is accomplished by standard collection procedures well known in the chemical and biochemical arts. In the context of the present invention recovering the multivalent protein preferably comprises collection of eluate fractions containing the peak of interest from either the cation exchange column, or the gel filtration HPLC column. Manual and automated fraction collection are well-known to one of ordinary skill in the art. Subsequent processing may involve lyophilization of the eluate to produce a stable solid, or further purification.

A third preferred method of producing multivalent antigen-binding proteins is to start with purified single-chain proteins at a lower concentration, and then increase the concentration until some significant fraction of multivalent proteins is formed. The multivalent proteins are then separated and recovered. The concentrations conducive to formation of multivalent proteins in this manner are from about 0.5 milligram per milliliter (mg/ml) to the concentration at which precipitates begin to form.

The use of the term "substantially free" when used to describe a composition of multivalent and single-chain antigen-binding protein molecules means the lack of a significant peak corresponding to the single-chain molecule, when the composition is analyzed by cation exchange chromatography, as disclosed in Example 1 or by gel filtration chromatography as disclosed in Example 2.

By use of the term "aqueous composition" is meant any composition of single-chain molecules and multivalent proteins including a portion of water. In the same context, the phrase "an excess of multivalent antigen-binding protein over single-chain molecules" indicates that the composition comprises more than 50% of multivalent antigen-binding protein.

The use of the term "cross-linking" refers to chemical means by which one can produce multivalent antigen-binding proteins from monovalent single-chain protein molecules. For example, the incorporation of a cross-linkable sulfhydryl chemical group as a cysteine residue in the single-chain proteins allows cross-linking by mild reduction of the sulfhydryl group. Both monospecific and multispecific multivalent proteins can be produced from single-chain proteins by cross-linking the free cysteine groups from two or more single-chain proteins, causing a covalent chemical linkage to form between the individual proteins. Free cysteines have been engineered into the C-terminal portion of the 4-4-20/212 single-chain antigen-binding protein, as discussed in Example 5 and Example 8. These free cysteines may then be cross-linked to form multivalent antigen-binding proteins.

The invention also comprises single-chain proteins, comprising: (a) a first polypeptide comprising the binding portion of the variable region of an antibody light chain; (b) a second polypeptide comprising the binding portion of the variable region of an antibody light chain; and (c) a peptide linker linking said first and second polypeptides (a) and (b) into said single-chain protein. A similar single-chain protein comprising the heavy chain variable regions is also a part of this invention. Genetic sequences encoding these molecules are also included in the scope of this invention. Since these proteins are comprised of two similar variable regions, they do not necessarily have any antigen-binding capability.

The invention also includes a DNA sequence encoding a bispecific bivalent antigen-binding protein. Example 4 and Example 7 discusses in detail the sequences that appear in FIGS. 10A and 10B that allow one of ordinary skill to construct a heterobivalent antigen-binding molecule. FIG. 10A is an amino acid and nucleotide sequence listing of the single-chain protein comprising the 4-4-20 V_(L) region connected through the 212 linker polypeptide to the CC49 V_(H) region. FIG. 10B is a similar listing of the single-hain protein comprising the CC49 V_(L) region connected through the 212 linker polypeptide to the 4-4-20 V_(H) region. Subjecting a composition including these single-chain molecules to dissociating and subsequent re-associating conditions results in the production of a bivalent protein with two different binding specificities.

Synthesis of DNA sequences is well know in the art, and possible through at least two routes. First, it is well-known that DNA sequences may be synthesized through the use of automated DNA synthesizers de novo, once the primary sequence information is known. Alternatively, it is possible to obtain a DNA sequence coding for a multivalent single-chain antigen-binding protein by removing the stop codons from the end of a gene encoding a single-chain antigen-binding protein, and then inserting a linker and a gene encoding a second single-chain antigen-binding protein. Example 6 demonstrates the construction of a DNA sequence coding for a bivalent single-chain antigen-binding protein. Other methods of genetically constructing multivalent single-chain antigen-binding proteins come within the spirit and scope of the present invention.

Having now generally described this invention the same will better be understood by reference to certain specific examples which are included for purposes of illustration and are not intended to limit it unless otherwise specified.

EXAMPLE 1 Production of Multivalent Antigen-Binding Proteins Dunng Purification

In the production of multivalent antigen-binding proteins, the same recombinant E. coli production system that was used for prior single-chain antigen-binding protein production was used. See Bird, et al., Science 242:423 (1988). This production system produced between 2 and 20% of the total E. coli protein as antigen-binding protein. For protein recovery, the frozen cell paste from three 10-liter fermentations (600-900 g) was thawed overnight at 4° C. and gently resuspended at 4° C. in 50 mM Tris-HCl, 1.0 mM EDTA, 100 mM KCl, 0.1 mM PMSF, pH 8.0 (lysis buffer), using 10 liters of lysis buffer for every kilogram of wet cell paste. When thoroughly resuspended, the chilled mixture was passed three times through a Manton-Gaulin cell homogenizer to totally lyse the cells. Because the cell homogenizer raised the temperature of the cell lysate to 25±5° C., the cell lysate was cooled to 5±2° C. with a Lauda/Brinkman chilling coil after each pass. Complete lysis was verified by visual inspection under a microscope.

The cell lysate was centrifuged at 24,300 g for 30 min. at 6° C. using a Sorvall RC-5B centrifuge. The pellet containing the insoluble antigen-binding protein was retained, and the supernatant was discarded. The pellet was washed by gently scraping it from the centrifuge bottles and resuspending it in 5 liters of lysis buffer/kg of wet cell paste. The resulting 3.0- to 4.5-liter suspension was again centrifuged at 24,300 g for 30 min at 6° C., and the supernatant was discarded. This washing of the pellet removes soluble E. coli proteins and can be repeated as many as five times. At any time during this washing procedure the material can be stored as a frozen pellet at -20° C. A substantial time saving in the washing steps can be accomplished by utilizing a Pellicon tangential flow apparatus equipped with 0.22-μm microporous filters, in place of centrifugation.

The washed pellet was solubilized at 4° C. in freshly prepared 6 M guanidine hydrochloride, 50 mM Tris-HCl, 10 mM CaCl₂, 50 mM KCl, pH 8.0 (dissociating buffer), using 9 ml/g of pellet. If necessary, a few quick pulses from a Heat Systems Ultrasonics tissue homogenizer can be used to complete the solubilization. The resulting suspension was centrifuged at 24,300 g for 45 min at 6° C. and the pellet was disca-ded. The optical density of the supernatant was determined at 280 nm and if the OD₂₈₀ was above 30, additional dissociating buffer was added to obtain an OD₂₈₀ of approximately 25.

The supernatant was slowly diluted into cold (4-7° C.) refolding buffer (50 mM Tris-HCl, 10 mM CaCl₂, 50 mM KCl, pH 8.0) until a 1:10 dilution was reached (final volume 10-20 liters). Re-folding occurs over approximately eighteen hours under these conditions. The best results are obtained when the GuHCl extract is slowly added to the refolding buffer over a 2-h period, with gentle mixing. The solution was left undisturbed for at least a 20-h period, and 95% ethanol was added to this solution such that the final ethanol concentration was approximately 20%. This solution was left undisturbed until the flocculated material settled to the bottom, usually not less than sixty minutes. The solution was filtered through a 0.2 um Millipore Millipak 200. This filtration step may be optionally preceded by a centrifugation step. The filtrate was concentrated to 1 to 2 liters using an Amicon spiral cartridge with a 10,000 MWCO cartridge, again at 4° C.

The concentrated crude antigen-binding protein sample was dialyzed against Buffer A (60 mM MOPS, 0.5 mM Ca acetate, pH 6.0-6.4) until the conductivity was lowered to that of Buffer A. The sample was then loaded on a 21.5×250-mm polyaspartic acid PolyCAT A column, manufactured by Poly LC of Columbia, Md. If more than 60 mg of protein is loaded on this column, the resolution begins to deteriorate; thus, the concentrated crude sample often must be divided into several PolyCAT A runs. Most antigen-binding proteins have an extinction coefficient of about 2.0 ml mg⁻¹ cm⁻¹ at 280 nm and this can be used to determine protein concentration. The antigen-binding protein sample was eluted from the PolyCAT A column with a 50-min linear gradient from Buffer A to Buffer B (see Table 1). Most of the single-chain proteins elute between 20 and 26 minutes when this gradient is used. This corresponds to an eluting solvent composition of approximately 70% Buffer A and 30% Buffer B. Most of the bivalent antigen-binding proteins elute later than 45 minutes, which correspond to over 90% Buffer B.

FIG. 7 is a chromatogram depicting the separation of single-chain protein from bivalent CC49/212 protein, using the cation-exchange method just described. Peak 1, 27.32 minutes, represents the monomeric single-chain fraction. Peak 2, 55.52 minutes, represents the bivalent protein fraction.

FIG. 8 is a chromatogram of the purified monomeric single-chain antigen-binding protein CC49/212 (Fraction 7 from FIG. 7) run on a Waters Protein-Pak 300SW gel filtration column. Monomer, with minor contaminates of dimer and trimer, is shown. FIG. 9 is a chromatogram of the purified bivalent antigen-binding protein CC49/212 (Fraction 15 from FIG. 7) run on the same Waters Protein-Pak 300SW gel filtration column as used in FIG. 8.

                  TABLE 1                                                          ______________________________________                                         PolyCAT A Cation-Exchange RPLC Gradients                                         Time         Flow     Buffers.sup.b                                          (min).sup.a                                                                               (ml/min) A          B    C                                          ______________________________________                                         Initial    15.0     100        0    0                                            50.0 15.0 0 100 0                                                              55.0 15.0 0 100 0                                                              60.0 15.0 0 0 100                                                              63.0 15.0 0 0 100                                                              64.0 15.0 100 0 0                                                              67.0 15.0 100 0 0                                                            ______________________________________                                          .sup.a Linear gradients are run between each time point.                       .sup.b Buffer A, 60 mM MOPS, 0.5 mM Ca acetate, pH 6.0-6.4;                    Buffer B, 60 mM MOPS, 20 mM Ca acetate, pH 7.5-8.0;                            Buffer C, 40 mM MOPS, 100 mM CaCl.sub.2, pH 7.5.                         

This purification procedure yielded multivalentantigen-binding proteins that are more than 95% pure as examined by SDS-PAGE and size exclusion HPLC. Modifications of the above procedure may be dictated by the isoelectric point of the particular multivalent antigen-binding protein being purified. Of the monomeric single-chain proteins that have been purified to date, all have had an isoelectric point (pI) between 8.0 and 9.5. However, it is possible that a multivalent antigen-binding protein may be produced with a pI of less than 7.0. In that case, an anion exchange column may be required for purification.

The CC49 monoclonal antibody was developed by Dr. Jeffrey Schlom's group, Laboratory of Tumor Immunology and Biology, National Cancer Institute. It binds specifically to the pan-carcinoma tumor antigen TAG-72. See Muraro, R. et al., Cancer Research 48:4588-4596 (1988).

To determine the binding properties of the bivalent and monomeric CC49/212 antigen-binding proteins, a competition radioimmunoassay (RIA) was set up in which a CC49 IgG (with two antigen binding sites) radiolabeled with ¹²⁵ I was competed against unlabeled CC49 IgG, or monovalent (fraction 7 in FIG. 7) or bivalent (fraction 15 in FIG. 7) CC49/212 antigen-binding protein for binding to the TAG-72 antigen on a human breast carcinoma extract. (See FIG. 18). This competition RIA showed that the bivalent antigen-binding protein competed equally well for the antigen as did IgG, whereas the monovalent single-chain antigen-binding protein needed a ten-fold higher protein concentration to displace the IgG. Thus, the monovalent antigen-binding protein competes with about a ten-fold lower affinity for the antigen than does the bivalent IgG or bivalent antigen-binding protein. FIG. 18 also shows the result of the competition RIA of a non-TAG-72 specific single-chain antigen-binding protein, the antifluorescein 4-4-20/212, which does not compete for binding.

EXAMPLE 2 Process of Making Multivalent Antigen-Binding Proteins Using Dissociating Agents

A. Process Using Guanidine HCl and Ethanol

Multivalent antigen-binding proteins were produced from purified single-chain proteins in the following way. First the purified single-chain protein at a concentration of 0.25-4 mg/ml was dialyzed against 0.5 moles/liter (M) guanidine hydrochloride (GuHCl), 20% ethanol (EtOH), in 0.05 M TRIS, 0.05 M KCl, 0.01 M CaCl₂ buffer pH 8.0. This combination of dissociating agents is thought to disrupt the V_(L) /V_(H) interface, allowing the V_(H) of a first single-chain molecule to come into contact with a V_(L) from a second single-chain molecule. Other dissociating agents such as urea, and alcohols such as isopropanol or methanol should be substitutable for GuHCl and EtOH. Following the initial dialysis, the protein was dialyzed against the load buffer for the final HPLC purification step. Two separate purification protocols, cation exchange and gel filtration chromatography, can be used to separate the single-chain protein monomer from the multivalent antigen-binding proteins. In the first method, monomeric and multivalent antigen-binding proteins were separated by using cation exchange HPLC chromography, using a polyaspartate column (PolyCAT A). This was a similar procedure to that used in the final purification of the antigen-binding proteins as described in Example 1. The load buffer was 0.06 M MOPS, 0.001 M Calcium Acetate pH 6.4. In the second method, the monomeric and multivalent antigen-binding proteins were separated by gel filtration HPLC chromatography using as a load buffer 0.04 M MOPS, 0.10 M Calcium Acetate pH 7.5. Gel filtration chromatography separates proteins based on their molecular size.

Once the antigen-binding protein sample wa:s loaded on the cation exchange HPLC column, a linear gradient was run between the load buffer (0.04 to 0.06 M MOPS, 0.000 to 0.001 M calcium acetate, 0 to 10% glycerol pH 6.0-6.4) and a second buffer (0.04 to 0.06 M MOPS, 0.01 to 0.02 M calcium acetate, 0 to 10% glycerol pH 7.5). It was important to have extensively dialyze the antigen-binding protein sample before loading it on the column. Normally, the conductivity of the sample is monitored against the dialysis buffer. Dialysis is continued until the conductivity drops below 600 μS. FIG. 11 shows the separation of the monomeric (27.83 min.) and bivalent (50.47 min.) forms of the CC49/212 antigen-binding protein by cation exchange. The chromatographic conditions for this separation were as follows: PolyCAT A column, 200×4.6 mm, operated at 0.62 ml/min.; load buffer and second buffer as in Example 1; gradient program from 100 percent load buffer A to 0 percent load buffer A over 48 mins; sample was CC49/212, 1.66 mg/ml; injection volume 0.2 ml. Fractions were collected from the two peaks from a similar chromatogram and identified as monomeric and bivalent proteins using gel filtration HPLC chromatography as described below.

Gel filtration HPLC chromatography (TSK G2000SW column from Toyo Soda, Tokyo, Japan) was used to identify and separate monomeric single-chain and multivalent antigen-binding proteins. This procedure has been described by Fukano, et al., J. Chromatography 166:47 (1978). Multimerization (creation of multivalent protein from monomeric single-chain protein) was by treatment with 0.5 M GuHCl and 20% EtOH for the times indicated in Table 2A followed by dialysis into the chromatography buffer. FIG. 12 shows the separation of monomeric (17.65 min.), bivalent (15.79 min.), trivalent (14.19 min.), and higher oligomers (shoulder at about 13.09 min.) of the B6.2/212 antigen-binding protein. The B6.2/212 single-chain antigen-binding protein is described in Colcher, D., et al., J. Nat. Cancer Inst. 82:1191-1197 (1990)). This separation depicts the results of a 24-hour multimerization treatment of a 1.0 mg/ml B6.2/212 antigen-binding protein sample. The HPLC buffer used was 0.04 M MOPS, 0.10 M calcium acetate, 0.04% sodium azide, pH 7.5.

FIG. 13 shows the results of a 24-hour treatment of a 4.0 mg/ml CC49/212 antigen-binding protein sample, generating monomeric, bivalent and trivalent proteins at 16.91, 14.9, and 13.42 min., respectively. The HPLC buffer was 40 mM MOPS, 100 mM calcium acetate, pH 7.35. Multimerization treatment was for the times indicated in Table 2.

The results of Example 2A are shown in Table 2A. Table 2A shows the percentage of bivalent and other multivalent forms before and after treatment with 20% ethanol and 0.5M GuHCl. Unless otherwise indicated, percentages were determined using a automatic data integration software package.

                  TABLE 2A                                                         ______________________________________                                         Summary of the generation of bivalent and higher                                 multivalent forms of B6.2/212 and CC49/212                                     proteins using guanidine hydrockloride and ethanol                                          Concen-                                                           Time tration %                                                               protein                                                                               (hours) (mg/ml)  monomer                                                                               dimer                                                                               trimer                                                                               multimers                            ______________________________________                                         CC49/212                                                                              0       0.25     86.7   11.6 1.7   0.0                                     0 1.0.sup.2 84.0 10.6 5.5 0.0                                                  0 4.0 70.0 17.1 12.9.sup.s 0.0                                                 2 0.25.sup.2 62.9 33.2 4.2 0.0                                                 2 1.0 24.2 70.6 5.1 0.0                                                        2 4.0 9.3 81.3 9.5 0.0                                                         26 0.25 16.0 77.6 6.4 0.0                                                      26 1.0 9.2 82.8 7.9 0.0                                                        26 4.0 3.7 78.2 18.1 0.0                                                      B6.2/212 0 0.25 100.0 0.0 0.0 0.0                                               0 1.0 100.0 0.0 0.0 0.0                                                        0 4.0 100.0 0.0 0.0 0.0                                                        2 0.25.sup.2 98.1 1.9 0.0 0.0                                                  2 1.0 100.0 0.0 0.0 0.0                                                        2 4.0 90.0 5.5 1.0 0.0                                                         24 0.25 45.6 37.5 10.2 6.7                                                     24 1.0 50.8 21.4 12.3 15.0                                                     24 4.0 5.9 37.2 25.7 29.9                                                   ______________________________________                                          .sup.1 Based on cut out peaks that were weighted.                              .sup.2 Average of two experiments.                                       

B. Process Using Urea and Ethanol

Multivalent antigen-binding proteins were produced from purified single-chain proteins in the following way. First the purified single-chain protein at a concentration of 0.25-1 mg/ml was dialyzed against 2M urea, 20% ethanol (EtOH), and 50 mM Tris buffer pH 8.0, for the times indicated in Table 2B. This combination of dissociating agents is thought to disrupt the V_(L) /V_(H) interface, allowing the V_(H) of a first single-chain molecule to come into contact with a V_(L) from a second single-chain molecule. Other dissociating agents such as isopropanol or methanol should be substitutable for EtOH. Following the initial dialysis, the protein was dialyzed against the load buffer for the final HPLC purification step.

Gel filtration HPLC chromatography (TSK G2000SW column from Toyo Soda, Tokyo, Japan) was used to identify and separate monomeric single-chain and multivalent antigen-binding proteins. This procedure has been described by Fukano, et al., J. Chromatography 166:47 (1978).

The results of Example 2B are shown in Table 2B. Table 2B shows the percentage of bivalent and other multivalent forms before and after treatment with 20% ethanol and urea. Percentages were determined using an automatic data integration software package.

                  TABLE 2B                                                         ______________________________________                                         Summary of the generation of bivalent and higher                                 multivalent forms of B6.2/212 and CC49/212                                     proteins using urea and ethanol                                                             Concen-                                                           Time tration %                                                               protein                                                                               (hours) (mg/ml)  monomer                                                                               dimer                                                                               trimer                                                                               multimers                            ______________________________________                                         B6.2   0       0.25     44.1   37.6 15.9  2.4                                     0 1.0 37.7 33.7 19.4 9.4                                                       3 0.25 22.2 66.5 11.3 0.0                                                      3 1.0 13.7 69.9 16.4 0.0                                                    ______________________________________                                    

EXAMPLE 3 Determination of Binding Constants

Three anti-fluorescein single-chain antigen-binding proteins have been constructed based on-the anti-fluorescein monoclonal antibody 4-4-20. The three 4-4-20 single-chain antigen-binding proteins differ in the polypeptide linker connecting the V_(H) and V_(L) regions of the protein. The three linkers used were 202', 212 and 216 (see Table 3). Bivalent and higher forms of the 4-4-20 antigen-binding protein were produced by concentrating the purified monomeric single-chain antigen-binding protein in the cation exchange load buffer (0.06 M MOPS, 0.001 M calcium acetate pH 6.4) to 5 mg/ml. The bivalent and monomeric forms of the 4-4-20 antigen-binding proteins were separated by cation exchange HPLC (polyaspartate column) using a 50 min. linear gradient between the load buffer (0.06 M MOPS, 0.001 M calcium acetate pH 6.4) and a second buffer (0.06 M MOPS, 0.02 M calcium acetate pH 7.5). Two 0.02 ml samples were separated, and fractions of the bivalent and monomeric protein peaks were collected on each run. The amount of protein contained in each fraction was determined from the absorbance at 278 nm from the first separation. Before collecting the fractions from the second separation run, each fraction tube had a sufficient quantity of 1.03×10⁻⁵ M fluorescein added to it, such that after the fractions were collected a 1-to-1 molar ratio of protein-to-fluorescein existed. Addition of fluorescein stabilized the bivalent form of the 4-4-20 antigen-binding proteins. These samples were kept at 2° C. (on ice).

The fluorescein dissociation rates were determined for each of these samples following the procedures described by Herron, J. N., in Fluorescence Hapten: An Immunological Probe, E. W. Voss, Ed., CRC Press, Boca Raton, Fla. (1984). A sample was first diluted with 20 mM HEPES buffer pH 8.0 to 5.0×10⁻⁸ M 4-4-20 antigen-binding protein. 560 μl of the 5.0×10⁻⁸ M 4-4-20 antigen-binding protein sample was added to a cuvette in a fluorescence spectrophotometer equilibrated at 2° C. and the fluorescence was read. 140 μl of 1.02×10⁻⁵ M fluoresceinamine was added to the cuvette, and the fluorescence was read every 1 minute for up to 25 minutes (see Table 4).

The binding constants (K_(a)) for the 4-4-20 single-chain antigen-binding protein monomers diluted in 20 mM HEPES buffer pH 8.0 in the absence of fluorescein were also determined (see Table 4).

The three polypeptide linkers in these experiments differ in length. The 202', 212 and 216 linkers are 12, 14 and 18 residues long, respectively. These experiments show that there are two effects of linker length on the 4-4-20 antigen-binding proteins: first, the shorter the linker length the higher the fraction of bivalent protein formed; second, the fluorescein dissociation rates of the monomeric single-chain antigen-binding proteins are effected more by the linker length than are the dissociation rates of the bivalent antigen-binding proteins. With the shorter linkers 202' and 212, the bivalent antigen-binding proteins have slower dissociation rates than the monomers. Thus, the linkers providing optimum production and binding affinities for monomeric and bivalent antigen-binding proteins may be different. Longer linkers may be more suitable for monomeric single-chain antigen-binding proteins, and shorter linkers may be more suitable for multivalent antigen-binding proteins.

                  TABLE 3                                                          ______________________________________                                         Linker Designs                                                                                                 Linker                                           V.sub.L Linker V.sub.H Name Reference                                        ______________________________________                                         KLEIE                                                                          GKSSGSGSESKS.sup.i                                                                               TQKLD-   202'   Bird                                               et al.                                                                      - -KLEIK GSTSGSGKSSEGKG.sup.2 EVKLD- 212 Bedzyk                                   et al.                                                                      - -KLEIK GSTSGSGKSSEGSGSTKG.sup.3 EVKLD- 216 This                                 application                                                                 - -KLVLK GSTSGKPSEGKG.sup.4 EVKLD- 217 This                                       application                                                              ______________________________________                                          (1) SEQ ID NO. 1                                                               (2) SEQ ID NO. 2                                                               (3) SEQ ID NO. 3                                                               (4) SEQ ID NO. 4                                                         

                  TABLE 4                                                          ______________________________________                                         Effects of Linkers on the SCA Protein Monomers and Dimers                                  Linker                                                                             202'       212      216                                        ______________________________________                                         Monomer                                                                          Fraction 0.47 0.66 0.90                                                        Ka 0.5 × 10.sup.9 M.sup.-1 1.0 × 10.sup.9 M.sup.-1 1.3                                             × 10.sup.9 M.sup.-1                      Dissociation rate 8.2 × 10.sup.-3 S.sup.-1 4.9 × 10.sup.-3                                         S.sup.-1 3.3 × 10.sup.-3 S.sup.-1        Dimer                                                                          Fraction 0.53 0.34 0.10                                                        Dissociation rate 4.6 × 10.sup.-3 S.sup.-1 3.5 × 10.sup.-3                                         S.sup.-1 3.5 × 10.sup.-3 S.sup.-1        Monomer/Dimer                                                                  Dissociation rate ratio 1.8 1.4 0.9                                          ______________________________________                                    

EXAMPLE 4 Genetic Construction of a Mixed-Fragment Bivalent Antigen-Binding Protein

The genetic constructions for one particular heterobivalent antigen-binding protein according to the Rearrangement model are shown in FIGS. 10A and 10B. FIG. 10A is an amino acid and nucleotide sequence listing of the 4-4-20 V_(L) /212/CC49 V_(H) construct, coding for a single-chain protein with a 4-4-20 V_(L), linked via a 212 polypeptide linker to a CC49 V_(H). FIG. 10B is a similar listing showing the CC49 V_(L) /212/4-4-20 V_(H) construct, coding for a single-chain protein with a CC49 V_(L), linked via a 212 linker to a 4-4-20 V_(H). These single-chain proteins may recombine according to the Rearrangement model to generate a heterobivalent protein comprising a CC49 antigen-binding site linked to a 4-4-20 antigen-binding site, as shown in FIG. 5B.

"4-4-20 V_(L) " means the variable region of the light chain of the 4-4-20 mouse monoclonal antibody (Bird, R. E. et al., Science 242:423 (1988)). The number "212" refers to a specific 14-residue polypeptide linker that links the 4-4-20 V_(L) and the CC49 V_(H). See Bedzyk, W. D. et al., J. Biol. Chem. 265:18615-18620 (1990). "CC49 V_(H) " is the variable region of the heavy chain of the CC49 antibody, which binds to the TAG-72 antigen. The CC49 antibody was developed at The National Institutes of Health by Schlom, et al. Generation and Charactefizadon of B72.3 Second Generation Monoclonal Antibodies Reactive With The Tumor-associated Glycoprotein 72 Antigen, Cancer Research 48:4588-4596 (1988).

Insertion of the sequences shown in FIGS. 10A and 10B, by standard recombinant DNA methodology, into a suitable plasmid vector will enable one of ordinary skill in the art to transform a suitable host for subsequent expression of the single-chain proteins. See Maniatis et al., Molecular Cloning, A Laboratory Manual, p. 104, Cold Spring Harbor Laboratory (1982), for general recombinant techniques for accomplishing the aforesaid goals; see also U.S. Pat. No. 4,946,778 (Ladner et al.) for a complete description of methods of producing single-chain protein molecules by recombinant DNA technology.

To produce multivalent antigen-binding proteins from the two single-chain proteins, 4-4-20V_(L) 212/CC49V_(H) and CC49V_(L) /212/4-4-20V_(H), the two single-hain proteins are dialyzed into 0.5 M GuHCl/20% EtOH being combined in a single solution either before or after dialysis. The multivalent proteins are then produced and separated as described in Example 2.

EXAMPLE 5 Preparation of Multivalent Antigen-Binding Proteins by Chemical Cross-Linking

Free cysteines were engineered into the C-terminal end of the 4-4-20/212 single-chain antigen-binding protein, in order to chemically crosslink the protein. The design was based on the hinge region found in antibodies between the C_(H) 1 and C_(H) 2 regions. In order to try to reduce antigenicity in humans, the hinge sequence of the most common IgG class, IgG1, was chosen. The 4-4-20 Fab structure was examined and it was determined that the C-terminal sequence GluH216-ProH217-ArgH218, was part of the C_(H) 1 region and that the hinge between C_(H) 1 and C_(H) 2 starts with ArgH218 or GlyH219 in the mouse 4-4-20 IgG2A antibody. FIG. 14 shows the structure of a human IgG. The hinge region is indicated generally. Thus the hinge from human IgG1 would start with LysH218 or SerH219. (See Table 5).

The C-terminal residue in most of the single-chain antigen-binding proteins described to date is the amino acid serine. In the design for the hinge region, the C-terminal serine in the 4-4-20/212 single-chain antigen-binding protein was made the first serine of the hinge and the second residue of the hinge was changed from a cysteine to a serine. This hinge cysteine normally forms a disulfide bridge to the C-terminal cysteine in the light chain.

                                      TABLE 5                                      __________________________________________________________________________                        218                                                                   |                                                             IgG2A mouse.sup.1   E P R G P T I K P     C P P C L C -                         - IgG1 human.sup.2 A E P K   S C D K T H T C P P C -                           - SCA*.sup.3                                                                  - V T V S                                                                       - SCA* Hinge design 1.sup.4                                                   - V T V S S D K T H T C                                                         - SCA* Hinge design 2.sup.5                                                   - V T V S S D K T H T C P P C                                                __________________________________________________________________________      *  singlechain antigenbinding protein                                          (1) SEQ ID NO. 5                                                               (2) SEQ ID NO. 6                                                               (3) SEQ ID NO. 7                                                               (4) SEQ ID NO. 8                                                               (5) SEQ ID NO. 9                                                         

There are possible advantages to having two C-terminal cysteines, for they might form an intramolecular disulfide bond, making the protein recovery easier by protecting the sulfurs from oxidation. The hinge regions were added by introduction of a BstE II restriction site in the 3'-terminus of the gene encoding the 4-4-20/212 single-chainantigen-binding protein (see FIGS. 15A-15B).

The monomeric single-chain antigen-binding protein containing the C-terminal cysteine can be purified using the normal methods of purifying a single-chain antigen-binding proteins, with minor modifications to protect the free sulfhydryls. The cross-linking could be accomplished in one of two ways. First, the purified single-chain antigen-binding protein could be treated with a mild reducing agent, such as dithiothreitol, then allowed to air oxidize to form a disulfide-bond between the individual single-chain antigen-binding proteins. This type of chemistry has been successful in producing heterodimers from whole antibodies (Nisonoff et al, Quantitative Estimation of the Hybridization of Rabbit Antibodies, Nature 4826:355-359 (1962); Brennan et al., Preparation of Bispecific Antibodies by Chemical Recombination of Monoclonal Immunoglobulin G₁ Fragments, Science 229:81-83 (1985)). Second, chemical crosslinking agents such as bismaleimidehexane could be used to cross-link two single-chain antigen-binding proteins by their C-terminal cysteines. See Partis et al., J. Prot. Chem. 2:263-277 (1983).

EXAMPLE 6 Genetic Construction of Bivalent Antigen-Binding Proteins

Bivalent antigen-binding proteins can be constructed genetically and subsequently expressed in E. coli or other known expression systems. This can be accomplished by genetically removing the stop codons at the end of a gene encoding a monomeric single-chain antigen-binding protein and inserting a linker and a gene encoding a second single-hain antigen-binding protein. We have constructed a gene for a bivalent CC49/212 antigen-binding protein in this manner (se FIG. 16). The CC49/212 gene in the starting expression plasmid is in an Aat II to Bam H1 restriction fragment (see Bird et al., Single-Chain Antigen-Binding Proteins, Science 242:423-426 (1988); and Whitlow et al., Single-Chain F_(V) Proteins and Their Fusion Proteins, Methods 2:97-105 (1991)). The two stop codons and the Bam H1 site at the C-terminal end of the CC49/212 antigen-binding protein gene were replaced by a single residue linker (Ser) and an Aat II restriction site. The resulting plasmid was cut with Aat II and the purified Aat II to Aat II restriction fragment was ligated into Aat II cut CC49/212 single-chain antigen-binding protein expression plasmid. The resulting bivalent CC49/212 single-chain antigen-binding protein expression plasmid was transfected into an E. coli expression host that contained the gene for the cI857 temperature-sensitive repressor. Expression of single-chain antigen-binding protein in this system is induced by raising the temperature from 30° C. to 42° C. FIG. 17 shows the expression of the divalent CC49/212 single-chain antigen-binding protein of FIG. 16 at 42° C., on an SDS-PAGE gel containing total E. coli protein. Lane 1 contains the molecular weight standards. Lane 2 is the uninduced E. coli production strain grown at 30° C. Lane 3 is divalent CC49/212 single-chain antigen-binding protein induced by growth at 42° C. The arrow shows the band of expressed divalent CC49/212 single-chain antigen-binding protein.

EXAMPLE 7 Construction, Purification, and Testing of 4-4-20/CC49 Heterodimer F_(V) With 217 Linkers

The goals of this experiment were to produce, purify and analyze for activity a new heterodimer F_(V) that would bind to both fluorescein and the pan-carcinoma antigen TAG-72. The design consisted of two polypeptide chains, which associated to form the active heterodimer Fv. Each polypeptide chain can be described as a mixed single-chain Fv (mixed sFv). The first mixed sFv (GX 8952) comprised a 4-4-20 variable light chain (V_(L)) and a CC49 variable heavy chain (V_(H)) connected by a 217 polypeptide linker (FIG. 19A). The second mixed sFv (GX 8953) comprised a CC-49 V_(L) and a 4-4-20 V_(H) connected by a 217 polypeptide linker (FIG. 19B). The sequence of the 217 polypeptide linker is shown in Table 3. Construction of analogous CC49/4-4-20 heterodimers connected by a 212 polypeptide linker were described in Example 4.

RESULTS

A. Purification

One 10-liter fermentation of each mixed sFv was grown on casein digest-glucose-salts medium at 32° C. to an optical density at 600 nm of 15 to 20. The mixed sFv expression was induced by raising the temperature of the fermentation to 42° C. for one hour. 277 gm (wet cell weight) of E. coli strain GX 8952 and 233 gm (wet cell weight) of E. coli strain GX 8953 were harvested in a centrifuge at 7000 g for 10 minutes. The cell pellets were kept and the supernate discarded. The cell pellets were frozen at -20° ° C. for storage.

2.55 liters of "lysis/wash buffer" (50 mM Tris/2 mM NaCl/1 mM EDTA, pH 8.0) was added to both of the mixed sFv's cell pellets, which were previously thawed and combined to give 510 gm of total wet cell weight. After complete suspension of the cells they were then passed through a Gaulin homogenizer at 9000 psi and 4° C. After this first pass the temperature increased to 23° C. The temperature was immediately brought down to 0° C. using dry ice and methanol. The cell suspension was passed through the Gaulin homogenizer a second time and centrifuged at 8000 rpm with a Dupont GS-3 rotor for 60 minutes. The supernatant was discarded after centrifugation and the pellets resuspended in 2.5 liters of "lysis/wash buffer" at 4° C. This suspension was centrifuged for 45 minutes at 8000 rpm with the Dupont GS-3 rotor. The supernatant was again discarded and the pellet weighed. The pellet weight was 136.1 gm.

1300 ml of 6M Guanidine Hydrochloride/50 mM Tris/50 mM KCl/10 mM CaCl₂ pH 8.0 at 4° C. was added to the washed pellet. An overhead mixer was used to speed solubilization. After one hour of mixing, the heterodimer GuHCl extract was centrifuged for 45 minutes at 8000 rpm and the pellet was discarded. The 1425 ml of heterodimer Fv 6M GuHCl extract was slowly added (16 ml/min) to 14.1 liters of "Refold Buffer" (50 mM Tris/50 mM KCl/10 mM CaCl₂, pH 8.0) under constant mixing at 4° C. to give an approximate dilution of 1:10. Refolding took place overnight at 4° C.

After 17 hours of refolding the anti-fluorescein activity was checked by a 40% quenching assay, and the amount of active protein calculated. 150 mg total active heterodimer Fv was found by the 40% quench assay, assuming a 54,000 molecular weight.

4 liters of prechilled (4° C.) 190 proof ethanol was added to the 15 liters of refolded heterodimer with mixing for 3 hours. The mixture sat overnight at 4° C. A flocculent precipitate had settled to the bottom after this overnight treatment. The nearly clear solution was filtered through a Millipak-200 (0.22μ) filter so as to not disturb the precipitate. A 40% quench assay showed that 10% of the anti-fluorescein activity was recovered in the filtrate.

The filtered sample of heterodimer was dialyzed, using a Pellicon system containing 10,000 dalton MWCO membranes, with "dialysis buffer" 40 mM MOPS/0.5 mM Calcium Acetate (CaAc), pH 6.4 at 4° C. 20 liters of dialysis buffer was required before the conductivity of the retentate was equal to that of the dialysis buffer (˜500 μS). After dialysis the heterodimer sample was filtered through a Millipak-20 filter, 0.22μ. After this step a 40% quench assay showed there was 8.8 mg of active protein.

The crude heterodimer sample was loaded on a Poly CAT A cation exchange column at 20 ml/min. The column was previously equilibrated with 60 mM MOPS, 1 mM CaAc pH 6.4, at 4° C., (Buffer A). After loading, the column was washed with 150 ml of "Buffer A" at 15 ml/min. A 50 min linear gradient was performed at 15 ml/min using "Buffer A" and "Buffer B" (60 mM MOPS, 20 mM CaAc pH 7.5 at 4° C.). The gradient conditions are presented in Table 6. "Buffer C" comprises 60 mM MOPS, 100 mM CaCl₂, pH 7.5.

                  TABLE 6                                                          ______________________________________                                         Time     % A    % B         % C  Flow                                          ______________________________________                                          0:00    100.0  0.0         0.0  15 ml/min                                       50:00 0.0 100.0 0.0 15 ml/min                                                  52:00 0.0 100.0 0.0 15 ml/min                                                  54:00 0.0 0.0 100.0 15 ml/min                                                  58:00 0.0 0.0 100.0 15 ml/min                                                  60:00 100.0 0.0 0.0 15 ml/min                                                ______________________________________                                    

Approximately 50 ml fractions were collected and analyzed for activity, purity, and molecular weight by size-exclusion chromatography. The fractions were not collected by peaks, so contamination between peaks is likely. Fractions 3 through 7 were pooled (total volume--218 ml), concentrated to 50 ml and dialyzed against 4 liters of 60 mM MOPS, 0.5 mM CaAc pH 6.4 at 4° C. overnight. The dialyzed pool was filtered through a 0.22μ filter and checked for absorbance at 280 nm. The filtrate was loaded onto the PolyCAT A column, equilibrated with 60 mM MOPS, 1 mM CaAc pH 6.4 at 4° C., at a flow rate of 10 ml/min. Buffer B was changed to 60 mM MOPS, 10 mM CaAc pH 7.5 at 4° C. The gradient was run as in Table 6. The fractions were collected by peak and analyzed for activity, purity, and molecular weight. The chromatogram is shown in FIG. 20. Fraction identification and analysis is presented in Table 7.

                  TABLE 7                                                          ______________________________________                                         Fraction Analysis of the Heterodimer Fv protein                                  Fraction            Total Volume                                                                             HPLC-SE Elution Time                             No. A.sub.280 reading (ml) (min)                                             ______________________________________                                         2      0.161      36          20.525                                             3 0.067 40                                                                     4 0.033 40                                                                     5 0.178 45 19.133                                                              6 0.234 50 19.163                                                              7 0.069 50                                                                     8 0.055 40                                                                   ______________________________________                                    

Fractions 2 to 7 and the starting material were analyzed by SDS gel electrophoresis, 4-20%. A picture and description of the gel is presented in FIG. 21.

B. HPLC Size Exclusion Results

Fractions 2, 5, and 6 correspond to the three main peaks in FIG. 20 and therefore were chosen to be analyzed by HPLC size exclusion. Fraction 2 corresponds to the peak that runs at 21.775 minutes in the preparative purification (FIG. 20), and runs on the HPLC sizing column at 20.525 minutes, which is in the monomeric position (FIG. 22A). Fractions 5 and 6 (30.1 and 33.455 minutes, respectively, in FIG. 20) run on the HPLC sizing column (FIGS. 22B and 22C) at 19.133 and 19.163 minutes, respectively (see Table 7). Therefore, both of these peaks could be considered dimers. 40% Quenching assays were performed on all fractions of this purification. Only fraction 5 gave significant activity. 2.4 mg of active CC49 4-4-20 heterodimer Fv was recovered in fraction 5, based on the Scatchard analysis described below.

C. N-terminal Sequencing of the Fractions

The active heterodimer Fv fraction should contain both polypeptide chains. N-terminal sequence analysis showed that fractions 5 and 6 displayed N-terminal sequences consistent with the prescence of both CC49 and 4-4-20 polypeptides and fraction 2 displayed a single sequence corresponding to the CC49/212/4-4-20 polypeptide only. We believe that fraction 6 was contaminated by fraction 5 (see FIG. 20), since only fraction 5 had significant activity.

D. Anti-Fluorescein Activity by Scatchard Analysis

The fluorescein association constants (Ka) were determined for fractions 5 and 6 using the fluorescence quenching assay described by Herron, J. N., in Fluorescence Hapten: An Immunological Probe, E. W. Voss, ed., CRC Press, Boca Raton, Fla. (1984). Each sample was diluted to approximately 5.0×10⁻⁸ M with 20 mM HEPES buffer pH 8.0. 590 μl of the 5.0×10⁻⁸ M sample was added to a cuvette in a fluorescence spectrophotometer equilibrated at room temperature. In a second cuvette 590 μl of 20 mM HEPES buffer pH 8.0 was added. To each cuvette was added 10 μl of 3.0×10⁻⁷ M fluorescein in 20 mM HEPES buffer pH 8.0, and the fluorescence recorded. This is repeated until 140 μl of fluorescein had been added. The resulting Scatchard analysis for fraction 5 shows a binding constant of 1.16×10⁹ M⁻¹ for fraction #5 (see FIG. 23). This is very close to the 4-4-20/212 sFv constant of 1.1×10⁹ M⁻¹ (see Pantoliano et al., Biochemistry 30:10117-10125 (1991)). The R intercept on the Scatchard analysis represents the fraction of active material. For fraction 5, 61% of the material was active. The graph of the Scatchard analysis on fraction 6 shows a binding constant of 3.3×10⁸ M⁻¹ and 14% active. The activity that is present in fraction 6 is most likely contaminants from fraction 5.

E. Anti-TAG-72 Activity by Competition ELISA

The CC49 monoclonal antibody was developed by Dr. Jeffrey Schlom's group, Laboratory of Tumor Immunology and Biology, National Cancer Institute. It binds specifically to the pan-carcinoma tumor antigen TAG-72. See Muraro, R., et al., Cancer Research 48:4588-4596 (1988).

To determine the binding properties of the bivalent CC49/4-4-20 Fv (fraction 5) and the CC49/212 sFv, a competition enzyme-linked immunosorbent assay (ELISA) was set up in which a CC49 IgG labeled with biotin was competed against unlabeled CC49/4-4-20 Fv and the CC49/212 sFv for binding to TAG-72 on a human breast carcinoma extract (see FIG. 24). The amount of biotin-labeled CC49 IgG was determined using a preformed complex with avidin and biotin coupled to horse radish peroxidase and O-phenylenediamine dihydrochloride (OPD). The reaction was stopped with 4N H₂ SO₄ (sulfuric acid), after 10 min. and the optical density read at 490 nm. This competition ELISA showed that the bivalent CC49/4-4-20 Fv binds to the TAG-72 antigen. The CC49/44-20 Fv needed a two hundred-fold higher protein concentration to displace the IgG than the singlechain Fv.

EXAMPLE 8 Cross-Linking Antigen-Binding Dimers

We have chemically crosslinked dimers of 4-4-20/212 antigen-binding protein with the two cysteine C-terminal extension (4-4-20/212 CPPC single-chain antigen-binding protein) in two ways. In Example 5 we describe the design and genetic construction of the 4-4-20/212 CPPC single-chain antigen-binding protein (hinge design 2 in Table 5). FIG. 15B shows the nucleic acid and protein sequences of this protein. After purifying the 4-4-20/212 CPPC single-chain antigen-binding protein, using the methods described in Whitlow and Filpula, Meth. Enzmol. 2:97 (1991), dimers were formed by two methods. First, the free cysteines were mildly reduced with dithiothreitol (DTT) and then the disulfide-bonds between the two molecules were allowed to form by air oxidation. Second, the chemical crosslinker bis-maleimidehexane was used to produce dimers by crosslinking the free cysteines from two 4-4-20/212 CPPC single-chain antigen-binding proteins.

A 0.1 mg/ml solution of the 4-4-20/212 CPPC single-hain antigen-binding protein was mildly reduced using 1 mM DTT, 50 mM HEPES, 50 mM NaCl, 1 mM EDTA buffer pH 8.0 at 4° C. The samples were dialyzed against 50 mM HEPES, 50 mM NaCl, 1 mM EDTA buffer pH 8.0 at 4° C. overnight, to allow the oxidation of free sulfhydrals to intermolecular disulfide-bonds. FIG. 25 shows a non-reducing SDS-PAGE gel after the air oxidation; it shows that approximately 10% of the 4-4-20/212 CPPC protein formed dimers with molecular weights around 55,000 Daltons.

A 0.1 mg/ml solution of the 4-4-20/212 CPPC single-chain antigen-binding protein was treated with 2 mM bis-maleimidehexane. Unlike forming a disulfide-bond between two free cysteines in the previous example, the bis-maleimidehexane crosslinker material should be stable to reducing agents such as β-mercaptoethanol. FIG. 26 shows that approximately 5% of the treated material produced dimer with a molecular weight of 55,000 Daltons on a reducing SDS-PAGE gel (samples were treated with β-mercaptalethanol prior to being loaded on the gel). We further purified the bis-maleimidehexane treated 4-4-20/212 CPPC protein on PolyCAT A cation exchange column after the protein had been extensively dialyzed against buffer A. FIG. 26 shows that we were able to enhance the fraction containing the dimer to approximately 15%.

CONCLUSIONS

We have produced a heterodimer Fv from two complementary mixed sFv's which has been shown to have the size of a dimer of the sFv's. The N-terminal analysis has shown that the active heterodimer Fv contains two polypeptide chains. The heterodimer Fv has been shown to be active for both fluorescein and TAG-72 binding.

All publications cited herein are incorporated fully into this disclosure by reference.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention and the following claims. As examples, the steps of the preferred embodiment constitute only one form of carrying out the process in which the invention may be embodied.

    __________________________________________________________________________     #             SEQUENCE LISTING                                                    - -  - - (1) GENERAL INFORMATION:                                              - -    (iii) NUMBER OF SEQUENCES: 23                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 12 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: both                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                - - Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Ly - #s Ser                       1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 14 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: both                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                - - Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Gl - #u Gly Lys Gly              1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: both                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                - - Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Gl - #u Gly Ser Gly Ser Thr       1               5   - #                10  - #                15                - - Lys Gly                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 12 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: both                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                - - Gly Ser Thr Ser Gly Lys Pro Ser Glu Gly Ly - #s Gly                       1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 15 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: both                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                - - Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pr - #o Pro Cys Leu Cys           1               5   - #                10  - #                15                - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 15 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: both                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                - - Ala Glu Pro Lys Ser Cys Asp Lys Thr His Th - #r Cys Pro Pro Cys           1               5   - #                10  - #                15                - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 4 amino - #acids                                                   (B) TYPE: amino acid                                                           (D) TOPOLOGY: both                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                - - Val Thr Val Ser                                                           1                                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: both                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                - - Val Thr Val Ser Ser Asp Lys Thr His Thr Cy - #s                           1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 14 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: both                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                - - Val Thr Val Ser Ser Asp Lys Thr His Thr Cy - #s Pro Pro Cys               1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 731 base - #pairs                                                  (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: both                                                         (D) TOPOLOGY: both                                                    - -     (ix) FEATURE:                                                                   (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..720                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                               - - GAC GTC GTT ATG ACT CAG ACA CCA CTA TCA CT - #T CCT GTT AGT CTA GGT            48                                                                        Asp Val Val Met Thr Gln Thr Pro Leu Ser Le - #u Pro Val Ser Leu Gly              1               5 - #                 10 - #                 15               - - GAT CAA GCC TCC ATC TCT TGC AGA TCT AGT CA - #G AGC CTT GTA CAC AGT            96                                                                        Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gl - #n Ser Leu Val His Ser                         20     - #             25     - #             30                   - - AAT GGA AAC ACC TAT TTA CGT TGG TAC CTG CA - #G AAG CCA GGC CAG TCT           144                                                                        Asn Gly Asn Thr Tyr Leu Arg Trp Tyr Leu Gl - #n Lys Pro Gly Gln Ser                     35         - #         40         - #         45                       - - CCA AAG GTC CTG ATC TAC AAA GTT TCC AAC CG - #A TTT TCT GGG GTC CCA           192                                                                        Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Ar - #g Phe Ser Gly Val Pro                 50             - #     55             - #     60                           - - GAC AGG TTC AGT GGC AGT GGA TCA GGG ACA GA - #T TTC ACA CTC AAG ATC           240                                                                        Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr As - #p Phe Thr Leu Lys Ile             65                 - # 70                 - # 75                 - # 80        - - AGC AGA GTG GAG GCT GAG GAT CTG GGA GTT TA - #T TTC TGC TCT CAA AGT           288                                                                        Ser Arg Val Glu Ala Glu Asp Leu Gly Val Ty - #r Phe Cys Ser Gln Ser                             85 - #                 90 - #                 95               - - ACA CAT GTT CCG TGG ACG TTC GGT GGA GGC AC - #C AAG CTT GAA ATC AAA           336                                                                        Thr His Val Pro Trp Thr Phe Gly Gly Gly Th - #r Lys Leu Glu Ile Lys                        100      - #           105      - #           110                   - - GGT TCT ACC TCT GGT TCT GGT AAA TCC TCT GA - #A GGC AAA GGT CAG GTT           384                                                                        Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Gl - #u Gly Lys Gly Gln Val                    115          - #       120          - #       125                       - - CAG CTG CAG CAG TCT GAC GCT GAG TTG GTG AA - #A CCT GGG GCT TCA GTG           432                                                                        Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Ly - #s Pro Gly Ala Ser Val                130              - #   135              - #   140                           - - AAG ATT TCC TGC AAG GCT TCT GGC TAC ACC TT - #C ACT GAC CAT GCA ATT           480                                                                        Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Ph - #e Thr Asp His Ala Ile            145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - CAC TGG GTG AAA CAG AAC CCT GAA CAG GGC CT - #G GAA TGG ATT GGA         TAT      528                                                                     His Trp Val Lys Gln Asn Pro Glu Gln Gly Le - #u Glu Trp Ile Gly Tyr                           165  - #               170  - #               175               - - TTT TCT CCC GGA AAT GAT GAT TTT AAA TAC AA - #T GAG AGG TTC AAG GGC           576                                                                        Phe Ser Pro Gly Asn Asp Asp Phe Lys Tyr As - #n Glu Arg Phe Lys Gly                        180      - #           185      - #           190                   - - AAG GCC ACA CTG ACT GCA GAC AAA TCC TCC AG - #C ACT GCC TAC GTG CAG           624                                                                        Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Se - #r Thr Ala Tyr Val Gln                    195          - #       200          - #       205                       - - CTC AAC AGC CTG ACA TCT GAG GAT TCT GCA GT - #G TAT TTC TGT ACA AGA           672                                                                        Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Va - #l Tyr Phe Cys Thr Arg                210              - #   215              - #   220                           - - TCC CTG AAT ATG GCC TAC TGG GGT CAA GGA AC - #C TCA GTC ACC GTC TCC           720                                                                        Ser Leu Asn Met Ala Tyr Trp Gly Gln Gly Th - #r Ser Val Thr Val Ser            225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - TAATAGGATC C               - #                  - #                       - #      731                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 240 amino - #acids                                                 (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                               - - Asp Val Val Met Thr Gln Thr Pro Leu Ser Le - #u Pro Val Ser Leu Gly         1               5 - #                 10 - #                 15               - - Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gl - #n Ser Leu Val His Ser                    20     - #             25     - #             30                   - - Asn Gly Asn Thr Tyr Leu Arg Trp Tyr Leu Gl - #n Lys Pro Gly Gln Ser                35         - #         40         - #         45                       - - Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Ar - #g Phe Ser Gly Val Pro            50             - #     55             - #     60                           - - Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr As - #p Phe Thr Leu Lys Ile        65                 - # 70                 - # 75                 - # 80        - - Ser Arg Val Glu Ala Glu Asp Leu Gly Val Ty - #r Phe Cys Ser Gln Ser                        85 - #                 90 - #                 95               - - Thr His Val Pro Trp Thr Phe Gly Gly Gly Th - #r Lys Leu Glu Ile Lys                   100      - #           105      - #           110                   - - Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Gl - #u Gly Lys Gly Gln Val               115          - #       120          - #       125                       - - Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Ly - #s Pro Gly Ala Ser Val           130              - #   135              - #   140                           - - Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Ph - #e Thr Asp His Ala Ile       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - His Trp Val Lys Gln Asn Pro Glu Gln Gly Le - #u Glu Trp Ile Gly         Tyr                                                                                              165  - #               170  - #               175              - - Phe Ser Pro Gly Asn Asp Asp Phe Lys Tyr As - #n Glu Arg Phe Lys Gly                   180      - #           185      - #           190                   - - Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Se - #r Thr Ala Tyr Val Gln               195          - #       200          - #       205                       - - Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Va - #l Tyr Phe Cys Thr Arg           210              - #   215              - #   220                           - - Ser Leu Asn Met Ala Tyr Trp Gly Gln Gly Th - #r Ser Val Thr Val Ser       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 744 base - #pairs                                                  (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: both                                                         (D) TOPOLOGY: both                                                    - -     (ix) FEATURE:                                                                   (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..732                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                               - - GAC GTC GTG ATG TCA CAG TCT CCA TCC TCC CT - #A CCT GTG TCA GTT         GGC       48                                                                     Asp Val Val Met Ser Gln Ser Pro Ser Ser Le - #u Pro Val Ser Val Gly             1               5 - #                 10 - #                 15               - - GAG AAG GTT ACT TTG AGC TGC AAG TCC AGT CA - #G AGC CTT TTA TAT AGT            96                                                                        Glu Lys Val Thr Leu Ser Cys Lys Ser Ser Gl - #n Ser Leu Leu Tyr Ser                         20     - #             25     - #             30                   - - GGT AAT CAA AAG AAC TAC TTG GCC TGG TAC CA - #G CAG AAA CCA GGG CAG           144                                                                        Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gl - #n Gln Lys Pro Gly Gln                     35         - #         40         - #         45                       - - TCT CCT AAA CTG CTG ATT TAC TGG GCA TCC GC - #T AGG GAA TCT GGG GTC           192                                                                        Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Al - #a Arg Glu Ser Gly Val                 50             - #     55             - #     60                           - - CCT GAT CGC TTC ACA GGC AGT GGA TCT GGG AC - #A GAT TTC ACT CTC TCC           240                                                                        Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Th - #r Asp Phe Thr Leu Ser             65                 - # 70                 - # 75                 - # 80        - - ATC AGC AGT GTG AAG ACT GAA GAC CTG GCA GT - #T TAT TAC TGT CAG CAG           288                                                                        Ile Ser Ser Val Lys Thr Glu Asp Leu Ala Va - #l Tyr Tyr Cys Gln Gln                             85 - #                 90 - #                 95               - - TAT TAT AGC TAT CCC CTC ACG TTC GGT GCT GG - #G ACC AAG CTT GTG CTG           336                                                                        Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gl - #y Thr Lys Leu Val Leu                        100      - #           105      - #           110                   - - AAA GGC TCT ACT TCC GGT AGC GGC AAA TCT TC - #T GAA GGT AAA GGT GAA           384                                                                        Lys Gly Ser Thr Ser Gly Ser Gly Lys Ser Se - #r Glu Gly Lys Gly Glu                    115          - #       120          - #       125                       - - GTT AAA CTG GAT GAG ACT GGA GGA GGC TTG GT - #G CAA CCT GGG AGG CCC           432                                                                        Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Va - #l Gln Pro Gly Arg Pro                130              - #   135              - #   140                           - - ATG AAA CTC TCC TGT GTT GCC TCT GGA TTC AC - #T TTT AGT GAC TAC TGG           480                                                                        Met Lys Leu Ser Cys Val Ala Ser Gly Phe Th - #r Phe Ser Asp Tyr Trp            145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - ATG AAC TGG GTC CGC CAG TCT CCA GAG AAA GG - #A CTG GAG TGG GTA         GCA      528                                                                     Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gl - #y Leu Glu Trp Val Ala                           165  - #               170  - #               175               - - CAA ATT AGA AAC AAA CCT TAT AAT TAT GAA AC - #A TAT TAT TCA GAT TCT           576                                                                        Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Th - #r Tyr Tyr Ser Asp Ser                        180      - #           185      - #           190                   - - GTG AAA GGC AGA TTC ACC ATC TCA AGA GAT GA - #T TCC AAA AGT AGT GTC           624                                                                        Val Lys Gly Arg Phe Thr Ile Ser Arg Asp As - #p Ser Lys Ser Ser Val                    195          - #       200          - #       205                       - - TAC CTG CAA ATG AAC AAC TTA AGA GTT GAA GA - #C ATG GGT ATC TAT TAC           672                                                                        Tyr Leu Gln Met Asn Asn Leu Arg Val Glu As - #p Met Gly Ile Tyr Tyr                210              - #   215              - #   220                           - - TGT ACG GGT TCT TAC TAT GGT ATG GAC TAC TG - #G GGT CAA GGA ACC TCA           720                                                                        Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Tr - #p Gly Gln Gly Thr Ser            225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - GTC ACC GTC TCC TAATAAGGAT CC       - #                  - #                    744                                                                     Val Thr Val Ser                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 244 amino - #acids                                                 (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                               - - Asp Val Val Met Ser Gln Ser Pro Ser Ser Le - #u Pro Val Ser Val Gly         1               5 - #                 10 - #                 15               - - Glu Lys Val Thr Leu Ser Cys Lys Ser Ser Gl - #n Ser Leu Leu Tyr Ser                    20     - #             25     - #             30                   - - Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gl - #n Gln Lys Pro Gly Gln                35         - #         40         - #         45                       - - Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Al - #a Arg Glu Ser Gly Val            50             - #     55             - #     60                           - - Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Th - #r Asp Phe Thr Leu Ser        65                 - # 70                 - # 75                 - # 80        - - Ile Ser Ser Val Lys Thr Glu Asp Leu Ala Va - #l Tyr Tyr Cys Gln Gln                        85 - #                 90 - #                 95               - - Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gl - #y Thr Lys Leu Val Leu                   100      - #           105      - #           110                   - - Lys Gly Ser Thr Ser Gly Ser Gly Lys Ser Se - #r Glu Gly Lys Gly Glu               115          - #       120          - #       125                       - - Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Va - #l Gln Pro Gly Arg Pro           130              - #   135              - #   140                           - - Met Lys Leu Ser Cys Val Ala Ser Gly Phe Th - #r Phe Ser Asp Tyr Trp       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gl - #y Leu Glu Trp Val         Ala                                                                                              165  - #               170  - #               175              - - Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Th - #r Tyr Tyr Ser Asp Ser                   180      - #           185      - #           190                   - - Val Lys Gly Arg Phe Thr Ile Ser Arg Asp As - #p Ser Lys Ser Ser Val               195          - #       200          - #       205                       - - Tyr Leu Gln Met Asn Asn Leu Arg Val Glu As - #p Met Gly Ile Tyr Tyr           210              - #   215              - #   220                           - - Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Tr - #p Gly Gln Gly Thr Ser       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Val Thr Val Ser                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 761 base - #pairs                                                  (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: both                                                         (D) TOPOLOGY: both                                                    - -     (ix) FEATURE:                                                                   (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..750                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                               - - GAC GTC GTT ATG ACT CAG ACA CCA CTA TCA CT - #T CCT GTT AGT CTA         GGT       48                                                                     Asp Val Val Met Thr Gln Thr Pro Leu Ser Le - #u Pro Val Ser Leu Gly             1               5 - #                 10 - #                 15               - - GAT CAA GCC TCC ATC TCT TGC AGA TCT AGT CA - #G AGC CTT GTA CAC AGT            96                                                                        Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gl - #n Ser Leu Val His Ser                         20     - #             25     - #             30                   - - AAT GGA AAC ACC TAT TTA CGT TGG TAC CTG CA - #G AAG CCA GGC CAG TCT           144                                                                        Asn Gly Asn Thr Tyr Leu Arg Trp Tyr Leu Gl - #n Lys Pro Gly Gln Ser                     35         - #         40         - #         45                       - - CCA AAG GTC CTG ATC TAC AAA GTT TCC AAC CG - #A TTT TCT GGG GTC CCA           192                                                                        Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Ar - #g Phe Ser Gly Val Pro                 50             - #     55             - #     60                           - - GAC AGG TTC AGT GGC AGT GGA TCA GGG ACA GA - #T TTC ACA CTC AAG ATC           240                                                                        Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr As - #p Phe Thr Leu Lys Ile             65                 - # 70                 - # 75                 - # 80        - - AGC AGA GTG GAG GCT GAG GAT CTG GGA GTT TA - #T TTC TGC TCT CAA AGT           288                                                                        Ser Arg Val Glu Ala Glu Asp Leu Gly Val Ty - #r Phe Cys Ser Gln Ser                             85 - #                 90 - #                 95               - - ACA CAT GTT CCG TGG ACG TTC GGT GGA GGC AC - #C AAG CTT GAA ATC AAA           336                                                                        Thr His Val Pro Trp Thr Phe Gly Gly Gly Th - #r Lys Leu Glu Ile Lys                        100      - #           105      - #           110                   - - GGT TCT ACC TCT GGT TCT GGT AAA TCT TCT GA - #A GGT AAA GGT GAA GTT           384                                                                        Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Gl - #u Gly Lys Gly Glu Val                    115          - #       120          - #       125                       - - AAA CTG GAT GAG ACT GGA GGA GGC TTG GTG CA - #A CCT GGG AGG CCC ATG           432                                                                        Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gl - #n Pro Gly Arg Pro Met                130              - #   135              - #   140                           - - AAA CTC TCC TGT GTT GCC TCT GGA TTC ACT TT - #T AGT GAC TAC TGG ATG           480                                                                        Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Ph - #e Ser Asp Tyr Trp Met            145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - AAC TGG GTC CGC CAG TCT CCA GAG AAA GGA CT - #G GAG TGG GTA GCA         CAA      528                                                                     Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Le - #u Glu Trp Val Ala Gln                           165  - #               170  - #               175               - - ATT AGA AAC AAA CCT TAT AAT TAT GAA ACA TA - #T TAT TCA GAT TCT GTG           576                                                                        Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Ty - #r Tyr Ser Asp Ser Val                        180      - #           185      - #           190                   - - AAA GGC AGA TTC ACC ATC TCA AGA GAT GAT TC - #C AAA AGT AGT GTC TAC           624                                                                        Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Se - #r Lys Ser Ser Val Tyr                    195          - #       200          - #       205                       - - CTG CAA ATG AAC AAC TTA AGA GTT GAA GAC AT - #G GGT ATC TAT TAC TGT           672                                                                        Leu Gln Met Asn Asn Leu Arg Val Glu Asp Me - #t Gly Ile Tyr Tyr Cys                210              - #   215              - #   220                           - - ACG GGT TCT TAC TAT GGT ATG GAC TAC TGG GG - #T CAA GGA ACC TCG GTC           720                                                                        Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gl - #y Gln Gly Thr Ser Val            225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - ACC GTC TCC AGT GAT AAG ACC CAT ACA TGC TA - #ATAGGATC C                    - #  761                                                                    Thr Val Ser Ser Asp Lys Thr His Thr Cys                                                        245  - #               250                                      - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 250 amino - #acids                                                 (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                               - - Asp Val Val Met Thr Gln Thr Pro Leu Ser Le - #u Pro Val Ser Leu Gly         1               5 - #                 10 - #                 15               - - Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gl - #n Ser Leu Val His Ser                    20     - #             25     - #             30                   - - Asn Gly Asn Thr Tyr Leu Arg Trp Tyr Leu Gl - #n Lys Pro Gly Gln Ser                35         - #         40         - #         45                       - - Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Ar - #g Phe Ser Gly Val Pro            50             - #     55             - #     60                           - - Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr As - #p Phe Thr Leu Lys Ile        65                 - # 70                 - # 75                 - # 80        - - Ser Arg Val Glu Ala Glu Asp Leu Gly Val Ty - #r Phe Cys Ser Gln Ser                        85 - #                 90 - #                 95               - - Thr His Val Pro Trp Thr Phe Gly Gly Gly Th - #r Lys Leu Glu Ile Lys                   100      - #           105      - #           110                   - - Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Gl - #u Gly Lys Gly Glu Val               115          - #       120          - #       125                       - - Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gl - #n Pro Gly Arg Pro Met           130              - #   135              - #   140                           - - Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Ph - #e Ser Asp Tyr Trp Met       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Le - #u Glu Trp Val Ala         Gln                                                                                              165  - #               170  - #               175              - - Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Ty - #r Tyr Ser Asp Ser Val                   180      - #           185      - #           190                   - - Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Se - #r Lys Ser Ser Val Tyr               195          - #       200          - #       205                       - - Leu Gln Met Asn Asn Leu Arg Val Glu Asp Me - #t Gly Ile Tyr Tyr Cys           210              - #   215              - #   220                           - - Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gl - #y Gln Gly Thr Ser Val       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Thr Val Ser Ser Asp Lys Thr His Thr Cys                                                   245  - #               250                                      - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 770 base - #pairs                                                  (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: both                                                         (D) TOPOLOGY: both                                                    - -     (ix) FEATURE:                                                                   (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..759                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                               - - GAC GTC GTT ATG ACT CAG ACA CCA CTA TCA CT - #T CCT GTT AGT CTA         GGT       48                                                                     Asp Val Val Met Thr Gln Thr Pro Leu Ser Le - #u Pro Val Ser Leu Gly             1               5 - #                 10 - #                 15               - - GAT CAA GCC TCC ATC TCT TGC AGA TCT AGT CA - #G AGC CTT GTA CAC AGT            96                                                                        Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gl - #n Ser Leu Val His Ser                         20     - #             25     - #             30                   - - AAT GGA AAC ACC TAT TTA CGT TGG TAC CTG CA - #G AAG CCA GGC CAG TCT           144                                                                        Asn Gly Asn Thr Tyr Leu Arg Trp Tyr Leu Gl - #n Lys Pro Gly Gln Ser                     35         - #         40         - #         45                       - - CCA AAG GTC CTG ATC TAC AAA GTT TCC AAC CG - #A TTT TCT GGG GTC CCA           192                                                                        Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Ar - #g Phe Ser Gly Val Pro                 50             - #     55             - #     60                           - - GAC AGG TTC AGT GGC AGT GGA TCA GGG ACA GA - #T TTC ACA CTC AAG ATC           240                                                                        Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr As - #p Phe Thr Leu Lys Ile             65                 - # 70                 - # 75                 - # 80        - - AGC AGA GTG GAG GCT GAG GAT CTG GGA GTT TA - #T TTC TGC TCT CAA AGT           288                                                                        Ser Arg Val Glu Ala Glu Asp Leu Gly Val Ty - #r Phe Cys Ser Gln Ser                             85 - #                 90 - #                 95               - - ACA CAT GTT CCG TGG ACG TTC GGT GGA GGC AC - #C AAG CTT GAA ATC AAA           336                                                                        Thr His Val Pro Trp Thr Phe Gly Gly Gly Th - #r Lys Leu Glu Ile Lys                        100      - #           105      - #           110                   - - GGT TCT ACC TCT GGT TCT GGT AAA TCT TCT GA - #A GGT AAA GGT GAA GTT           384                                                                        Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Gl - #u Gly Lys Gly Glu Val                    115          - #       120          - #       125                       - - AAA CTG GAT GAG ACT GGA GGA GGC TTG GTG CA - #A CCT GGG AGG CCC ATG           432                                                                        Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gl - #n Pro Gly Arg Pro Met                130              - #   135              - #   140                           - - AAA CTC TCC TGT GTT GCC TCT GGA TTC ACT TT - #T AGT GAC TAC TGG ATG           480                                                                        Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Ph - #e Ser Asp Tyr Trp Met            145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - AAC TGG GTC CGC CAG TCT CCA GAG AAA GGA CT - #G GAG TGG GTA GCA         CAA      528                                                                     Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Le - #u Glu Trp Val Ala Gln                           165  - #               170  - #               175               - - ATT AGA AAC AAA CCT TAT AAT TAT GAA ACA TA - #T TAT TCA GAT TCT GTG           576                                                                        Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Ty - #r Tyr Ser Asp Ser Val                        180      - #           185      - #           190                   - - AAA GGC AGA TTC ACC ATC TCA AGA GAT GAT TC - #C AAA AGT AGT GTC TAC           624                                                                        Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Se - #r Lys Ser Ser Val Tyr                    195          - #       200          - #       205                       - - CTG CAA ATG AAC AAC TTA AGA GTT GAA GAC AT - #G GGT ATC TAT TAC TGT           672                                                                        Leu Gln Met Asn Asn Leu Arg Val Glu Asp Me - #t Gly Ile Tyr Tyr Cys                210              - #   215              - #   220                           - - ACG GGT TCT TAC TAT GGT ATG GAC TAC TGG GG - #T CAA GGA ACC TCG GTC           720                                                                        Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gl - #y Gln Gly Thr Ser Val            225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - ACC GTC TCC AGT GAT AAG ACC CAT ACA TGC CC - #T CCA TGC TAATAGGATC      C     770                                                                       Thr Val Ser Ser Asp Lys Thr His Thr Cys Pr - #o Pro Cys                                        245  - #               250                                      - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 253 amino - #acids                                                 (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                               - - Asp Val Val Met Thr Gln Thr Pro Leu Ser Le - #u Pro Val Ser Leu Gly         1               5 - #                 10 - #                 15               - - Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gl - #n Ser Leu Val His Ser                    20     - #             25     - #             30                   - - Asn Gly Asn Thr Tyr Leu Arg Trp Tyr Leu Gl - #n Lys Pro Gly Gln Ser                35         - #         40         - #         45                       - - Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Ar - #g Phe Ser Gly Val Pro            50             - #     55             - #     60                           - - Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr As - #p Phe Thr Leu Lys Ile        65                 - # 70                 - # 75                 - # 80        - - Ser Arg Val Glu Ala Glu Asp Leu Gly Val Ty - #r Phe Cys Ser Gln Ser                        85 - #                 90 - #                 95               - - Thr His Val Pro Trp Thr Phe Gly Gly Gly Th - #r Lys Leu Glu Ile Lys                   100      - #           105      - #           110                   - - Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Gl - #u Gly Lys Gly Glu Val               115          - #       120          - #       125                       - - Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gl - #n Pro Gly Arg Pro Met           130              - #   135              - #   140                           - - Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Ph - #e Ser Asp Tyr Trp Met       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Le - #u Glu Trp Val Ala         Gln                                                                                              165  - #               170  - #               175              - - Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Ty - #r Tyr Ser Asp Ser Val                   180      - #           185      - #           190                   - - Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Se - #r Lys Ser Ser Val Tyr               195          - #       200          - #       205                       - - Leu Gln Met Asn Asn Leu Arg Val Glu Asp Me - #t Gly Ile Tyr Tyr Cys           210              - #   215              - #   220                           - - Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gl - #y Gln Gly Thr Ser Val       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Thr Val Ser Ser Asp Lys Thr His Thr Cys Pr - #o Pro Cys                                   245  - #               250                                      - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1460 base - #pairs                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: both                                                         (D) TOPOLOGY: both                                                    - -     (ix) FEATURE:                                                                   (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..1449                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                               - - GAC GTC GTG ATG TCA CAG TCT CCA TCC TCC CT - #A CCT GTG TCA GTT         GGC       48                                                                     Asp Val Val Met Ser Gln Ser Pro Ser Ser Le - #u Pro Val Ser Val Gly             1               5 - #                 10 - #                 15               - - GAG AAG GTT ACT TTG AGC TGC AAG TCC AGT CA - #G AGC CTT TTA TAT AGT            96                                                                        Glu Lys Val Thr Leu Ser Cys Lys Ser Ser Gl - #n Ser Leu Leu Tyr Ser                         20     - #             25     - #             30                   - - GGT AAT CAA AAG AAC TAC TTG GCC TGG TAC CA - #G CAG AAA CCA GGG CAG           144                                                                        Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gl - #n Gln Lys Pro Gly Gln                     35         - #         40         - #         45                       - - TCT CCT AAA CTG CTG ATT TAC TGG GCA TCC GC - #T AGG GAA TCT GGG GTC           192                                                                        Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Al - #a Arg Glu Ser Gly Val                 50             - #     55             - #     60                           - - CCT GAT CGC TTC ACA GGC AGT GGA TCT GGG AC - #A GAT TTC ACT CTC TCC           240                                                                        Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Th - #r Asp Phe Thr Leu Ser             65                 - # 70                 - # 75                 - # 80        - - ATC AGC AGT GTG AAG ACT GAA GAC CTG GCA GT - #T TAT TAC TGT CAG CAG           288                                                                        Ile Ser Ser Val Lys Thr Glu Asp Leu Ala Va - #l Tyr Tyr Cys Gln Gln                             85 - #                 90 - #                 95               - - TAT TAT AGC TAT CCC CTC ACG TTC GGT GCT GG - #G ACC AAG CTT GTG CTG           336                                                                        Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gl - #y Thr Lys Leu Val Leu                        100      - #           105      - #           110                   - - AAA GGC TCT ACT TCC GGT AGC GGC AAA TCC TC - #T GAA GGC AAA GGT CAG           384                                                                        Lys Gly Ser Thr Ser Gly Ser Gly Lys Ser Se - #r Glu Gly Lys Gly Gln                    115          - #       120          - #       125                       - - GTT CAG CTG CAG CAG TCT GAC GCT GAG TTG GT - #G AAA CCT GGG GCT TCA           432                                                                        Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Va - #l Lys Pro Gly Ala Ser                130              - #   135              - #   140                           - - GTG AAG ATT TCC TGC AAG GCT TCT GGC TAC AC - #C TTC ACT GAC CAT GCA           480                                                                        Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Th - #r Phe Thr Asp His Ala            145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - ATT CAC TGG GTG AAA CAG AAC CCT GAA CAG GG - #C CTG GAA TGG ATT         GGA      528                                                                     Ile His Trp Val Lys Gln Asn Pro Glu Gln Gl - #y Leu Glu Trp Ile Gly                           165  - #               170  - #               175               - - TAT TTT TCT CCC GGA AAT GAT GAT TTT AAA TA - #C AAT GAG AGG TTC AAG           576                                                                        Tyr Phe Ser Pro Gly Asn Asp Asp Phe Lys Ty - #r Asn Glu Arg Phe Lys                        180      - #           185      - #           190                   - - GGC AAG GCC ACA CTG ACT GCA GAC AAA TCC TC - #C AGC ACT GCC TAC GTG           624                                                                        Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Se - #r Ser Thr Ala Tyr Val                    195          - #       200          - #       205                       - - CAG CTC AAC AGC CTG ACA TCT GAG GAT TCT GC - #A GTG TAT TTC TGT ACA           672                                                                        Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Al - #a Val Tyr Phe Cys Thr                210              - #   215              - #   220                           - - AGA TCC CTG AAT ATG GCC TAC TGG GGT CAA GG - #A ACC TCA GTC ACC GTC           720                                                                        Arg Ser Leu Asn Met Ala Tyr Trp Gly Gln Gl - #y Thr Ser Val Thr Val            225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - TCC TCA GAC GTC GTG ATG TCA CAG TCT CCA TC - #C TCC CTA CCT GTG         TCA      768                                                                     Ser Ser Asp Val Val Met Ser Gln Ser Pro Se - #r Ser Leu Pro Val Ser                           245  - #               250  - #               255               - - GTT GGC GAG AAG GTT ACT TTG AGC TGC AAG TC - #C AGT CAG AGC CTT TTA           816                                                                        Val Gly Glu Lys Val Thr Leu Ser Cys Lys Se - #r Ser Gln Ser Leu Leu                        260      - #           265      - #           270                   - - TAT AGT GGT AAT CAA AAG AAC TAC TTG GCC TG - #G TAC CAG CAG AAA CCA           864                                                                        Tyr Ser Gly Asn Gln Lys Asn Tyr Leu Ala Tr - #p Tyr Gln Gln Lys Pro                    275          - #       280          - #       285                       - - GGG CAG TCT CCT AAA CTG CTG ATT TAC TGG GC - #A TCC GCT AGG GAA TCT           912                                                                        Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Al - #a Ser Ala Arg Glu Ser                290              - #   295              - #   300                           - - GGG GTC CCT GAT CGC TTC ACA GGC AGT GGA TC - #T GGG ACA GAT TTC ACT           960                                                                        Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Se - #r Gly Thr Asp Phe Thr            305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - CTC TCC ATC AGC AGT GTG AAG ACT GAA GAC CT - #G GCA GTT TAT TAC         TGT     1008                                                                     Leu Ser Ile Ser Ser Val Lys Thr Glu Asp Le - #u Ala Val Tyr Tyr Cys                           325  - #               330  - #               335               - - CAG CAG TAT TAT AGC TAT CCC CTC ACG TTC GG - #T GCT GGG ACC AAG CTT          1056                                                                        Gln Gln Tyr Tyr Ser Tyr Pro Leu Thr Phe Gl - #y Ala Gly Thr Lys Leu                        340      - #           345      - #           350                   - - GTG CTG AAA GGC TCT ACT TCC GGT AGC GGC AA - #A TCC TCT GAA GGC AAA          1104                                                                        Val Leu Lys Gly Ser Thr Ser Gly Ser Gly Ly - #s Ser Ser Glu Gly Lys                    355          - #       360          - #       365                       - - GGT CAG GTT CAG CTG CAG CAG TCT GAC GCT GA - #G TTG GTG AAA CCT GGG          1152                                                                        Gly Gln Val Gln Leu Gln Gln Ser Asp Ala Gl - #u Leu Val Lys Pro Gly                370              - #   375              - #   380                           - - GCT TCA GTG AAG ATT TCC TGC AAG GCT TCT GG - #C TAC ACC TTC ACT GAC          1200                                                                        Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gl - #y Tyr Thr Phe Thr Asp            385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - CAT GCA ATT CAC TGG GTG AAA CAG AAC CCT GA - #A CAG GGC CTG GAA         TGG     1248                                                                     His Ala Ile His Trp Val Lys Gln Asn Pro Gl - #u Gln Gly Leu Glu Trp                           405  - #               410  - #               415               - - ATT GGA TAT TTT TCT CCC GGA AAT GAT GAT TT - #T AAA TAC AAT GAG AGG          1296                                                                        Ile Gly Tyr Phe Ser Pro Gly Asn Asp Asp Ph - #e Lys Tyr Asn Glu Arg                        420      - #           425      - #           430                   - - TTC AAG GGC AAG GCC ACA CTG ACT GCA GAC AA - #A TCC TCC AGC ACT GCC          1344                                                                        Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Ly - #s Ser Ser Ser Thr Ala                    435          - #       440          - #       445                       - - TAC GTG CAG CTC AAC AGC CTG ACA TCT GAG GA - #T TCT GCA GTG TAT TTC          1392                                                                        Tyr Val Gln Leu Asn Ser Leu Thr Ser Glu As - #p Ser Ala Val Tyr Phe                450              - #   455              - #   460                           - - TGT ACA AGA TCC CTG AAT ATG GCC TAC TGG GG - #T CAA GGA ACC TCA GTC          1440                                                                        Cys Thr Arg Ser Leu Asn Met Ala Tyr Trp Gl - #y Gln Gly Thr Ser Val            465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - ACC GTC TCC TAATAGGATC C         - #                  - #                      146 - #0                                                                 Thr Val Ser                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 483 amino - #acids                                                 (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                               - - Asp Val Val Met Ser Gln Ser Pro Ser Ser Le - #u Pro Val Ser Val Gly         1               5 - #                 10 - #                 15               - - Glu Lys Val Thr Leu Ser Cys Lys Ser Ser Gl - #n Ser Leu Leu Tyr Ser                    20     - #             25     - #             30                   - - Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gl - #n Gln Lys Pro Gly Gln                35         - #         40         - #         45                       - - Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Al - #a Arg Glu Ser Gly Val            50             - #     55             - #     60                           - - Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Th - #r Asp Phe Thr Leu Ser        65                 - # 70                 - # 75                 - # 80        - - Ile Ser Ser Val Lys Thr Glu Asp Leu Ala Va - #l Tyr Tyr Cys Gln Gln                        85 - #                 90 - #                 95               - - Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gl - #y Thr Lys Leu Val Leu                   100      - #           105      - #           110                   - - Lys Gly Ser Thr Ser Gly Ser Gly Lys Ser Se - #r Glu Gly Lys Gly Gln               115          - #       120          - #       125                       - - Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Va - #l Lys Pro Gly Ala Ser           130              - #   135              - #   140                           - - Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Th - #r Phe Thr Asp His Ala       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Ile His Trp Val Lys Gln Asn Pro Glu Gln Gl - #y Leu Glu Trp Ile         Gly                                                                                              165  - #               170  - #               175              - - Tyr Phe Ser Pro Gly Asn Asp Asp Phe Lys Ty - #r Asn Glu Arg Phe Lys                   180      - #           185      - #           190                   - - Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Se - #r Ser Thr Ala Tyr Val               195          - #       200          - #       205                       - - Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Al - #a Val Tyr Phe Cys Thr           210              - #   215              - #   220                           - - Arg Ser Leu Asn Met Ala Tyr Trp Gly Gln Gl - #y Thr Ser Val Thr Val       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Ser Ser Asp Val Val Met Ser Gln Ser Pro Se - #r Ser Leu Pro Val         Ser                                                                                              245  - #               250  - #               255              - - Val Gly Glu Lys Val Thr Leu Ser Cys Lys Se - #r Ser Gln Ser Leu Leu                   260      - #           265      - #           270                   - - Tyr Ser Gly Asn Gln Lys Asn Tyr Leu Ala Tr - #p Tyr Gln Gln Lys Pro               275          - #       280          - #       285                       - - Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Al - #a Ser Ala Arg Glu Ser           290              - #   295              - #   300                           - - Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Se - #r Gly Thr Asp Phe Thr       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Leu Ser Ile Ser Ser Val Lys Thr Glu Asp Le - #u Ala Val Tyr Tyr         Cys                                                                                              325  - #               330  - #               335              - - Gln Gln Tyr Tyr Ser Tyr Pro Leu Thr Phe Gl - #y Ala Gly Thr Lys Leu                   340      - #           345      - #           350                   - - Val Leu Lys Gly Ser Thr Ser Gly Ser Gly Ly - #s Ser Ser Glu Gly Lys               355          - #       360          - #       365                       - - Gly Gln Val Gln Leu Gln Gln Ser Asp Ala Gl - #u Leu Val Lys Pro Gly           370              - #   375              - #   380                           - - Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gl - #y Tyr Thr Phe Thr Asp       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - His Ala Ile His Trp Val Lys Gln Asn Pro Gl - #u Gln Gly Leu Glu         Trp                                                                                              405  - #               410  - #               415              - - Ile Gly Tyr Phe Ser Pro Gly Asn Asp Asp Ph - #e Lys Tyr Asn Glu Arg                   420      - #           425      - #           430                   - - Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Ly - #s Ser Ser Ser Thr Ala               435          - #       440          - #       445                       - - Tyr Val Gln Leu Asn Ser Leu Thr Ser Glu As - #p Ser Ala Val Tyr Phe           450              - #   455              - #   460                           - - Cys Thr Arg Ser Leu Asn Met Ala Tyr Trp Gl - #y Gln Gly Thr Ser Val       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Thr Val Ser                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 725 base - #pairs                                                  (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: both                                                         (D) TOPOLOGY: both                                                    - -     (ix) FEATURE:                                                                   (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..714                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                               - - GAC GTC GTT ATG ACT CAG ACA CCA CTA TCA CT - #T CCT GTT AGT CTA         GGT       48                                                                     Asp Val Val Met Thr Gln Thr Pro Leu Ser Le - #u Pro Val Ser Leu Gly             1               5 - #                 10 - #                 15               - - GAT CAA GCC TCC ATC TCT TGC AGA TCT AGT CA - #G AGC CTT GTA CAC AGT            96                                                                        Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gl - #n Ser Leu Val His Ser                         20     - #             25     - #             30                   - - AAT GGA AAC ACC TAT TTA CGT TGG TAC CTG CA - #G AAG CCA GGC CAG TCT           144                                                                        Asn Gly Asn Thr Tyr Leu Arg Trp Tyr Leu Gl - #n Lys Pro Gly Gln Ser                     35         - #         40         - #         45                       - - CCA AAG GTC CTG ATC TAC AAA GTT TCC AAC CG - #A TTT TCT GGG GTC CCA           192                                                                        Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Ar - #g Phe Ser Gly Val Pro                 50             - #     55             - #     60                           - - GAC AGG TTC AGT GGC AGT GGA TCA GGG ACA GA - #T TTC ACA CTC AAG ATC           240                                                                        Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr As - #p Phe Thr Leu Lys Ile             65                 - # 70                 - # 75                 - # 80        - - AGC AGA GTG GAG GCT GAG GAT CTG GGA GTT TA - #T TTC TGC TCT CAA AGT           288                                                                        Ser Arg Val Glu Ala Glu Asp Leu Gly Val Ty - #r Phe Cys Ser Gln Ser                             85 - #                 90 - #                 95               - - ACA CAT GTT CCG TGG ACG TTC GGT GGA GGC AC - #C AAG CTT GAA ATC AAA           336                                                                        Thr His Val Pro Trp Thr Phe Gly Gly Gly Th - #r Lys Leu Glu Ile Lys                        100      - #           105      - #           110                   - - GGT TCT ACC TCT GGT AAA CCA TCT GAA GGC AA - #A GGT CAG GTT CAG CTG           384                                                                        Gly Ser Thr Ser Gly Lys Pro Ser Glu Gly Ly - #s Gly Gln Val Gln Leu                    115          - #       120          - #       125                       - - CAG CAG TCT GAC GCT GAG TTG GTG AAA CCT GG - #G GCT TCA GTG AAG ATT           432                                                                        Gln Gln Ser Asp Ala Glu Leu Val Lys Pro Gl - #y Ala Ser Val Lys Ile                130              - #   135              - #   140                           - - TCC TGC AAG GCT TCT GGC TAC ACC TTC ACT GA - #C CAT GCA ATT CAC TGG           480                                                                        Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr As - #p His Ala Ile His Trp            145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - GTG AAA CAG AAC CCT GAA CAG GGC CTG GAA TG - #G ATT GGA TAT TTT         TCT      528                                                                     Val Lys Gln Asn Pro Glu Gln Gly Leu Glu Tr - #p Ile Gly Tyr Phe Ser                           165  - #               170  - #               175               - - CCC GGA AAT GAT GAT TTT AAA TAC AAT GAG AG - #G TTC AAG GGC AAG GCC           576                                                                        Pro Gly Asn Asp Asp Phe Lys Tyr Asn Glu Ar - #g Phe Lys Gly Lys Ala                        180      - #           185      - #           190                   - - ACA CTG ACT GCA GAC AAA TCC TCC AGC ACT GC - #C TAC GTG CAG CTC AAC           624                                                                        Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Al - #a Tyr Val Gln Leu Asn                    195          - #       200          - #       205                       - - AGC CTG ACA TCT GAG GAT TCT GCA GTG TAT TT - #C TGT ACA AGA TCC CTG           672                                                                        Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Ph - #e Cys Thr Arg Ser Leu                210              - #   215              - #   220                           - - AAT ATG GCC TAC TGG GGT CAA GGA ACC TCA GT - #C ACC GTC TCC              TAATAGGATC   724                                                                 Asn Met Ala Tyr Trp Gly Gln Gly Thr Ser Va - #l Thr Val Ser                    225                 2 - #30                 2 - #35                             - - C                  - #                  - #                  - #                   725                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 238 amino - #acids                                                 (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                               - - Asp Val Val Met Thr Gln Thr Pro Leu Ser Le - #u Pro Val Ser Leu Gly         1               5 - #                 10 - #                 15               - - Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gl - #n Ser Leu Val His Ser                    20     - #             25     - #             30                   - - Asn Gly Asn Thr Tyr Leu Arg Trp Tyr Leu Gl - #n Lys Pro Gly Gln Ser                35         - #         40         - #         45                       - - Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Ar - #g Phe Ser Gly Val Pro            50             - #     55             - #     60                           - - Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr As - #p Phe Thr Leu Lys Ile        65                 - # 70                 - # 75                 - # 80        - - Ser Arg Val Glu Ala Glu Asp Leu Gly Val Ty - #r Phe Cys Ser Gln Ser                        85 - #                 90 - #                 95               - - Thr His Val Pro Trp Thr Phe Gly Gly Gly Th - #r Lys Leu Glu Ile Lys                   100      - #           105      - #           110                   - - Gly Ser Thr Ser Gly Lys Pro Ser Glu Gly Ly - #s Gly Gln Val Gln Leu               115          - #       120          - #       125                       - - Gln Gln Ser Asp Ala Glu Leu Val Lys Pro Gl - #y Ala Ser Val Lys Ile           130              - #   135              - #   140                           - - Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr As - #p His Ala Ile His Trp       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Val Lys Gln Asn Pro Glu Gln Gly Leu Glu Tr - #p Ile Gly Tyr Phe         Ser                                                                                              165  - #               170  - #               175              - - Pro Gly Asn Asp Asp Phe Lys Tyr Asn Glu Ar - #g Phe Lys Gly Lys Ala                   180      - #           185      - #           190                   - - Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Al - #a Tyr Val Gln Leu Asn               195          - #       200          - #       205                       - - Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Ph - #e Cys Thr Arg Ser Leu           210              - #   215              - #   220                           - - Asn Met Ala Tyr Trp Gly Gln Gly Thr Ser Va - #l Thr Val Ser               225                 2 - #30                 2 - #35                             - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 738 base - #pairs                                                  (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: both                                                         (D) TOPOLOGY: both                                                    - -     (ix) FEATURE:                                                                   (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..726                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                               - - GAC GTC GTG ATG TCA CAG TCT CCA TCC TCC CT - #A CCT GTG TCA GTT GGC            48                                                                        Asp Val Val Met Ser Gln Ser Pro Ser Ser Le - #u Pro Val Ser Val Gly              1               5 - #                 10 - #                 15               - - GAG AAG GTT ACT TTG AGC TGC AAG TCC AGT CA - #G AGC CTT TTA TAT AGT            96                                                                        Glu Lys Val Thr Leu Ser Cys Lys Ser Ser Gl - #n Ser Leu Leu Tyr Ser                         20     - #             25     - #             30                   - - GGT AAT CAA AAG AAC TAC TTG GCC TGG TAC CA - #G CAG AAA CCA GGG CAG           144                                                                        Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gl - #n Gln Lys Pro Gly Gln                     35         - #         40         - #         45                       - - TCT CCT AAA CTG CTG ATT TAC TGG GCA TCC GC - #T AGG GAA TCT GGG GTC           192                                                                        Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Al - #a Arg Glu Ser Gly Val                 50             - #     55             - #     60                           - - CCT GAT CGC TTC ACA GGC AGT GGA TCT GGG AC - #A GAT TTC ACT CTC TCC           240                                                                        Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Th - #r Asp Phe Thr Leu Ser             65                 - # 70                 - # 75                 - # 80        - - ATC AGC AGT GTG AAG ACT GAA GAC CTG GCA GT - #T TAT TAC TGT CAG CAG           288                                                                        Ile Ser Ser Val Lys Thr Glu Asp Leu Ala Va - #l Tyr Tyr Cys Gln Gln                             85 - #                 90 - #                 95               - - TAT TAT AGC TAT CCC CTC ACG TTC GGT GCT GG - #G ACC AAG CTT GTG CTG           336                                                                        Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gl - #y Thr Lys Leu Val Leu                        100      - #           105      - #           110                   - - AAA GGC TCT ACT TCC GGT AAA CCA TCT GAA GG - #T AAA GGT GAA GTT AAA           384                                                                        Lys Gly Ser Thr Ser Gly Lys Pro Ser Glu Gl - #y Lys Gly Glu Val Lys                    115          - #       120          - #       125                       - - CTG GAT GAG ACT GGA GGA GGC TTG GTG CAA CC - #T GGG AGG CCC ATG AAA           432                                                                        Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pr - #o Gly Arg Pro Met Lys                130              - #   135              - #   140                           - - CTC TCC TGT GTT GCC TCT GGA TTC ACT TTT AG - #T GAC TAC TGG ATG AAC           480                                                                        Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Se - #r Asp Tyr Trp Met Asn            145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - TGG GTC CGC CAG TCT CCA GAG AAA GGA CTG GA - #G TGG GTA GCA CAA         ATT      528                                                                     Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Gl - #u Trp Val Ala Gln Ile                           165  - #               170  - #               175               - - AGA AAC AAA CCT TAT AAT TAT GAA ACA TAT TA - #T TCA GAT TCT GTG AAA           576                                                                        Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Ty - #r Ser Asp Ser Val Lys                        180      - #           185      - #           190                   - - GGC AGA TTC ACC ATC TCA AGA GAT GAT TCC AA - #A AGT AGT GTC TAC CTG           624                                                                        Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Ly - #s Ser Ser Val Tyr Leu                    195          - #       200          - #       205                       - - CAA ATG AAC AAC TTA AGA GTT GAA GAC ATG GG - #T ATC TAT TAC TGT ACG           672                                                                        Gln Met Asn Asn Leu Arg Val Glu Asp Met Gl - #y Ile Tyr Tyr Cys Thr                210              - #   215              - #   220                           - - GGT TCT TAC TAT GGT ATG GAC TAC TGG GGT CA - #A GGA ACC TCA GTC ACC           720                                                                        Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gl - #n Gly Thr Ser Val Thr            225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - GTC TCC TAATAAGGAT CC           - #                  - #                       - # 738                                                                  Val Ser                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 242 amino - #acids                                                 (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                               - - Asp Val Val Met Ser Gln Ser Pro Ser Ser Le - #u Pro Val Ser Val Gly         1               5 - #                 10 - #                 15               - - Glu Lys Val Thr Leu Ser Cys Lys Ser Ser Gl - #n Ser Leu Leu Tyr Ser                    20     - #             25     - #             30                   - - Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gl - #n Gln Lys Pro Gly Gln                35         - #         40         - #         45                       - - Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Al - #a Arg Glu Ser Gly Val            50             - #     55             - #     60                           - - Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Th - #r Asp Phe Thr Leu Ser        65                 - # 70                 - # 75                 - # 80        - - Ile Ser Ser Val Lys Thr Glu Asp Leu Ala Va - #l Tyr Tyr Cys Gln Gln                        85 - #                 90 - #                 95               - - Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gl - #y Thr Lys Leu Val Leu                   100      - #           105      - #           110                   - - Lys Gly Ser Thr Ser Gly Lys Pro Ser Glu Gl - #y Lys Gly Glu Val Lys               115          - #       120          - #       125                       - - Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pr - #o Gly Arg Pro Met Lys           130              - #   135              - #   140                           - - Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Se - #r Asp Tyr Trp Met Asn       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Gl - #u Trp Val Ala Gln         Ile                                                                                              165  - #               170  - #               175              - - Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Ty - #r Ser Asp Ser Val Lys                   180      - #           185      - #           190                   - - Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Ly - #s Ser Ser Val Tyr Leu               195          - #       200          - #       205                       - - Gln Met Asn Asn Leu Arg Val Glu Asp Met Gl - #y Ile Tyr Tyr Cys Thr           210              - #   215              - #   220                           - - Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gl - #n Gly Thr Ser Val Thr       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Val Ser                                                                 __________________________________________________________________________ 

What is claimed is:
 1. A method of producing a multivalent antigen-binding protein comprising the steps of:(a) producing a composition comprising single-chain molecules, each single-chain molecule comprising:(i) a first polypeptide comprising the binding portion of the variable region of an antibody light chain; (ii) a second polypeptide comprising the binding portion of the variable region of an antibody heavy chain; and (iii) a peptide linker linking said first and second polypentides (i) and (ii); or (iv) a first polypeptide comprising the binding portion of the variable region of an antibody heavy chain; (v) a second polypeptide comprising the binding portion of the variable region of an antibody light chain; and (vi) a peptide linker linking said first and second polypeptides (iv) and (v); (b) dissociating said single-chain molecules; (c) re-associating said single-chain molecules; (d) separating multivalent antigen-binding proteins from said single-chain molecules; and (e) recovering said multivalent proteins.
 2. The method of claim 1 wherein said dissociation is caused by dialysis against a dissociating solution.
 3. The method of claim 1 wherein said reassociation is caused by dialysis against a refolding solution or a refolding agent.
 4. A method of producing a multivalent antigen-binding protein, comprising the step of cross-linking at least two single-chain molecules to each other, each single-chain molecule comprising:(a) first polypeptide comprising the binding portion of the variable region of an antibody light chain; (b) a second polypeptide comprising the binding portion of the variable region of an antibody heavy chain; and (c) a peptide linker linking said first and second polypeptides (a) and (b); or (d) a first polypeptide comprising the binding portion of the variable region of an antibody heavy chain; (e) a second polypeptide comprising the binding portion of the variable region of an antibody light chain; and (f) a peptide linker linking said first and second polypeptides (d) and (e).
 5. The method of claim 4 wherein said cross-linking is effected by chemical means.
 6. The method of claim 2 wherein said dissociating agent is gunldine hydrochloride.
 7. The method of claim 2 wherein said dissociating agent is urea.
 8. The method of claim 2 wherein said dissociating agent is an alcohol.
 9. The method of claim 8 wherein said alcohol is ethanol.
 10. A method of producing a multivalent antigen-binding protein, comprising the steps of:(a) producing a composition comprising multivalent antigen-binding protein and single-chain molecules, each single-chain molecule comprising:(i) a first polypeptide comprising the binding portion of the variable region of an antibody light chain; (ii) a second polypeptide comprising the binding portion of the variable region of an antibody heavy chain; and (iii) a peptide linker linking said first and second polypeptides (i) and (ii); or (iv) a first polypeptide comprising the binding portion of the variable region of an antibody heavy chain; (v) a second polypeptide comprising the binding portion of the variable region of an antibody light chain; and (vi) a peptide linker linking said first and second polypeptides (iv) and (v), (b) concentrating said single-chain molecules; (c) separating said multivalent protein from said single-chain molecules; and (d) recovering said multivalent protein.
 11. The method of claim 10 wherein said concentrating step occurs from 0.5 mg/ml single-chain molecule to the concentration at which precipitation starts.
 12. A method of producing a multivalent antigen-binding protein, comprising the steps of:(a) producing a composition comprising multivalent antigen-binding protein and single-chain molecules, each single-chain molecule comprising:(i) a first polypeptide comprising the binding portion of the variable region of an antibody light chain; (ii) a second polypeptide comprising the binding portion of the variable region of an antibody heavy chain; and (iii) a peptide linker linking said first and second polypeptides (i) and (ii); or (iv) a first polypeptide comprising the binding portion of the variable region of an antibody heavy chain; (v) a second polypeptide comprising the binding portion of the variable region of an antibody light chain; and (vi) a peptide linker linking said first and second polypeptides (iv) and (v), (b) separating said multivalent protein from said single-chain molecules; and (c) recovering said multivalent protein.
 13. The method of claim 12 wherein separating said multivalent protein from said single-chain molecules comprises utilizing cation exchange chromatography.
 14. The method of claim 12 wherein separating said multivalent protein from said single-chain molecules comprises utilizing gel filtration chromatography.
 15. A method of producing a multivalent antigen-binding protein comprising two or more single-chain molecules, each single-chain molecule comprising:(a) a first polypeptide comprising the binding portion of the variable region of an antibody light chain; (b) a second polypeptide comprising the binding portion of the variable region of an antibody heavy chain; and (c) a peptide linker linking said first and second polypeptides (a) and (b); or (d) a first polypeptide comprising the binding portion of the variable region of an antibody heavy chain; (e) a second polypeptide comprising the binding portion of the variable region of an antibody light chain; and (f) a peptide linker linking said first and second polypeptides (d) and (e), said method comprising:(i) providing a genetic sequence coding for said single-chain molecule; (ii) transforming one or more host cells with said sequence; (iii) expressing said sequence in said host or hosts; and (iv) recovering said multivalent protein from said host or hosts.
 16. The method of claim 15 wherein recovering said multivalent protein comprises separating said multivalent protein from said single-chain molecules.
 17. The method of claim 15 wherein recovering said multivalent protein comprises:(a) dissociating said single-chain molecules; (b) re-associating said single-chain molecules; (c) separating multivalent antigen-binding proteins from said single-chain molecules; and (d) recovering said multivalent proteins.
 18. The method of claim 15 which further comprises purifying said recovered multivalent protein.
 19. The method of claim 15 wherein said host cell is a bacterial cell, a yeast cell or other fungal cell, or a mammalian cell line.
 20. The method of claim 19 wherein said host cell is Escherichia coli or Bacillus subtilis.
 21. A method of producing a composition comprising an antigen-binding protein having more than 50% of said protein in multivalent form, said protein in multivalent form comprising two or more single-chain molecules, each single-chain molecule comprising:(a) a first polypeptide comprising the binding portion of the variable region of an antibody light chain; (b) a second polypeptide comprising the binding portion of the variable region of an antibody heavy chain; and (c) a peptide linker linking said first and second polypeptides (a) and (b); or (d) a first polypeptide comprising the binding portion of the variable region of an antibody heavy chain; (e) a second polypeptide comprising the binding portion of the variable region of an antibody light chain; and (f) a peptide linker linking said first and second polypeptides (d) and (e), said method comprising:(i) dissociating said single-chain molecules; (ii) re-associating said single-chain molecules; (iii) separating multivalent antigen-binding proteins from said single-chain molecules; and (iv) recovering said multivalent proteins.
 22. The method of claim 21, said composition having more than 84% of said protein in multivalent form.
 23. The method of claim 21, said composition having more than 95% of said protein in multivalent form.
 24. The method of claim 21 wherein a dissociating agent is used to dissociate said single-chain molecules.
 25. The method of claim 24 wherein said dissociating agent is guanidine hydrochloride.
 26. The method of claim 24 wherein said dissociating agent is urea. 